Compositions for inhibiting checkpoint gene expression and uses thereof

ABSTRACT

The present invention is directed to compounds, compositions, and methods useful for modulating PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA or protein expression using gene silencing compounds comprising two or more single stranded antisense oligonucleotides that are linked through their 5′-ends to allow the presence of two or more accessible 3′-ends.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional patent application Ser. No. 62/126,368, filed on Feb. 27, 2015, the contents of which are incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention relates to compounds, compositions, and methods of use for the inhibition of checkpoint gene expression or for diagnosing, treating and/or preventing diseases and/or conditions that respond to the inhibition of checkpoint gene expression.

Summary of the Related Art

The immune system is a hosts defense against foreign antigens; however, in order to function properly a variety of checks and balances are required to protect against self-antigens (i.e., autoimmunity) and, at the same time, provide an appropriate response against foreign. Immune-activating and immune-suppressive receptors and ligands provide these regulatory checks and balances (see Pardoll et al., The blockade of immune checkpoints in cancer immunotherapy, Nat. Rev. Canc. 12, 252 (2012)).

Immune checkpoints refer to a group of endogenous immune-suppressive ligands and receptors that are crucial for the maintenance of self-tolerance and the protection of tissues from damage when the immune system is responding to an infection. (see Y. L. Wu, et al., Immunotherapies: The Blockade of Inhibitory Signals, Int. J. Biol. Sci. 8, 1420 (2012)) In response to the induction of an immune response expression of checkpoints increases. These checkpoints act as regulatory feedback to maintain immune homeostasis.

In patients with cancer, tumor mutations give rise to tumor-specific antigens that can be recognized by the immune system, particularly T-cells, leading to elimination of cancer cells. However, to defend themselves, tumor cells can co-opt immune checkpoint pathways to suppress the immune response in the tumor microenvironment and evade the host immune system by inhibiting T cells that might otherwise attack the tumor cells. (see J. F. Grosso & M. N. Jure-Kunkel; CTLA-4 blockade in tumor models: an overview of preclinical and translational research, Cancer Immun. 13, 5 (2013); M. E. Turnis, et al.; Combinatorial immunotherapy: PD-1 may not be LAG-ing behind any more, OncoImmunology 1, 1172 (2012)).

Many previous cancer immunotherapies have likely been limited by these suppressive mechanisms. Thus there is a need to over these immunosuppressive mechanisms in order to enhance antitumor immunotherapy applications.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to compounds, compositions, and methods useful for modulating PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA or protein expression using gene silencing compounds comprising two or more single stranded antisense oligonucleotides that are linked through their 5′-ends to allow the presence of two or more accessible 3′-ends. The gene silencing compounds according to the invention effectively inhibit or decrease PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA or protein expression.

Provided herein are methods, compounds, and compositions for modulating expression of PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA and protein. In certain embodiments, compounds useful for modulating expression of PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA and protein are gene silencing compounds.

In certain embodiments, modulation can occur in a cell or tissue. In certain embodiments the cell is a tumor cell. In certain embodiments, the tissue is a tumor. In certain embodiments, the cell or tissue is in an animal. In certain embodiments, the animal is a human. In certain embodiments, PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA levels are reduced. In certain embodiments, PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L protein levels are reduced. Such reduction can occur in a time-dependent manner or in a dose-dependent manner.

Also provided are methods, compounds, and compositions useful for preventing, treating, and ameliorating diseases, disorders, and conditions. In certain embodiments, such diseases, disorders, and conditions are hyperproliferative diseases, disorders, and conditions. In certain embodiments such hyperproliferative diseases, disorders, and conditions include cancer as well as associated malignancies and metastases.

In certain embodiments, methods of treatment include administering a PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L gene silencing compound or composition to an individual in need thereof. In certain embodiments, the gene silencing compound or composition is administered intratumorally.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The invention relates to the therapeutic and prophylactic use of gene silencing compounds, also referred to as 3^(rd) generation antisense (3GA) compounds, to down-regulate checkpoint mRNA or protein expression. Such molecules are useful, for example, in providing compositions for modulation of checkpoint gene expression or for treating and/or preventing diseases and/or conditions that are capable of responding to modulation of checkpoint gene expression in patients, subjects, animals or organisms.

The objects of the present invention, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the accompanying drawings in which the following terms have the ascribed meaning. Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.

The term “2′-O-substituted” means substitution of the 2′ position of the pentose moiety with an -O-lower alkyl group containing 1-6 saturated or unsaturated carbon atoms (for example, but not limited to, 2′-O-methyl), or with an -O-aryl or allyl group having 2-6 carbon atoms, wherein such alkyl, aryl or allyl group may be unsubstituted or may be substituted, (for example, with 2′-O-methoxyethyl, ethoxy, methoxy, halo, hydroxyl, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxyl, carbalkoxyl, or amino groups); or with a hydroxyl, an amino or a halo group, but not with a 2′-H group. In some embodiments the oligonucleotides of the invention include four or five 2′-O-alky nucleotides at their 5′ terminus, and/or four or five 2′-O-alky nucleotides at their 3′ terminus.

The term “3”, when used directionally, generally refers to a region or position in a polynucleotide or oligonucleotide 3′ (toward the 3′end of the nucleotide) from another region or position in the same polynucleotide or oligonucleotide.

The term “3′ end” generally refers to the 3′ terminal nucleotide of the component oligonucleotides. “Two or more oligonucleotides linked at their 3′ ends” generally refers to a linkage between the 3′ terminal nucleotides of the oligonucleotides which may be directly via 5′, 3′ or 2′ hydroxyl groups, or indirectly, via a non-nucleotide linker. Such linkages may also be via a nucleoside, utilizing both 2′ and 3′ hydroxyl positions of the nucleoside. Such linkages may also utilize a functionalized sugar or nucleobase of a 3′terminal nucleotide.

The term “5′”, when used directionally, generally refers to a region or position in a polynucleotide or oligonucleotide 5′ (toward the 5′end of the nucleotide) from another region or position in the same polynucleotide or oligonucleotide.

The term “5′ end” generally refers to the 5′ terminal nucleotide of the component oligonucleotides. “Two or more single-stranded antisense oligonucleotides linked at their 5′ ends” generally refers to a linkage between the 5′ terminal nucleotides of the oligonucleotides which may be directly via 5′, 3′ or 2′ hydroxyl groups, or indirectly, via a non-nucleotide linker. Such linkages may also be via a nucleoside, utilizing both 2′ and 3′ hydroxyl positions of the nucleoside. Such linkages may also utilize a functionalized sugar or nucleobase of a 5′terminal nucleotide.

The term “about” generally means that the exact number is not critical. Thus, oligonucleotides having one or two fewer nucleoside residues, or from one to several additional nucleoside residues are contemplated as equivalents of each of the embodiments described above.

The term “accessible” generally means when related to a compound according to the invention, that the relevant portion of the molecule is able to be recognized by the cellular components necessary to elicit an intended response to the compound.

The term “agonist” generally refers to a substance that binds to a receptor of a cell and induces a response. An agonist can be a naturally occurring substance such as bacterial DNA or a synthetic composition. A synthetic agonist often mimics the action of a naturally occurring substance such as a ligand.

The term “antigen” generally refers to a substance that is recognized and selectively bound by an antibody or by a T cell antigen receptor. Antigens may include but are not limited to peptides, proteins, lipids, carbohydrates, nucleosides, nucleotides, nucleic acids, and combinations thereof. Antigens may be natural or synthetic and generally induce an immune response that is specific for that antigen.

“Antisense activity” means any detectable or measurable activity attributable to the hybridization of a gene silencing compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.

As used herein, “Gene silencing oligonucleotide (GSO)”, “Gene silencing compound”, or “3^(rd) generation antisense (3GA)” compound are used interchangeably to refer to an oligomeric compound comprising two or more single stranded antisense oligonucleotides that are linked through their 5′-ends to allow the presence of two or more accessible 3′-ends. Gene silencing compounds are capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

“Antisense inhibition” means reduction of target nucleic acid levels or target protein levels in the presence of a gene silencing compound complementary to a target nucleic acid as compared to target nucleic acid levels or target protein levels in the absence of the gene silencing compound.

“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.

The term “biologic instability” generally refers to a molecule's ability to be degraded and subsequently inactivated in vivo. For oligonucleotides, such degradation results from exonuclease activity and/or endonuclease activity, wherein exonuclease activity refers to cleaving nucleotides from the 3′ or 5′ end of an oligonucleotide, and endonuclease activity refers to cleaving phosphodiester bonds at positions other than at the ends of the oligonucleotide.

The term “cancer” generally refers to, without limitation, any malignant growth or tumor caused by abnormal or uncontrolled cell proliferation and/or division. Cancers may occur in humans and/or mammals and may arise in any and all tissues. Treating a patient having cancer may include administration of a compound, pharmaceutical formulation or vaccine according to the invention such that the abnormal or uncontrolled cell proliferation and/or division, or metastasis is affected.

The term “carrier” generally encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid, lipid containing vesicle, microspheres, liposomal encapsulation, or other material for use in pharmaceutical formulations. It will be understood that the characteristics of the carrier, excipient or diluent will depend on the route of administration for a particular application. The preparation of pharmaceutically acceptable formulations containing these materials is described in, for example, Remington's Pharmaceutical Sciences, 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, Pa., 1990.

The term “co-administration” or “co-administered” generally refers to the administration of at least two different substances. Co-administration refers to simultaneous administration, as well as temporally spaced order of up to several days apart, of at least two different substances in any order, either in a single dose or separate doses.

The term “in combination with” generally means administering two or more agents (e.g., a gene silencing compound according to the invention and another agent) such that there is an overlap of an effect of each agent on the patient. Such administration may be done in any order, including simultaneous administration, as well as temporally spaced order from a few seconds up to several days apart. In some embodiments, the administration of the agents are spaced sufficiently close together such that a combinatorial effect is achieved. Such combination treatment may also include more than a single administration of the compound according to the invention and/or independently the other agent. The administration of the compound according to the invention and the other agent may be by the same or different routes. In some embodiments, administration of at least one agent is made while the other agent is still present at a therapeutic level in the subject.

The term “complementary” is intended to mean the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.

“Contiguous nucleobases” means nucleobases immediately adjacent to each other.

The term “individual” or “subject” or “patient” generally refers to a mammal, such as a human.

“CEACAM1 nucleic acid” means any nucleic acid encoding CEACAM1. For example, in certain embodiments, a CEACAM1 nucleic acid includes a DNA sequence encoding CEACAM1, an RNA sequence transcribed from DNA encoding CEACAM1 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding CEACAM1. “CEACAM1 mRNA” means an mRNA encoding a CEACAM1 protein.

“CTLA4 nucleic acid” means any nucleic acid encoding CTLA4. For example, in certain embodiments, a CTLA4 nucleic acid includes a DNA sequence encoding CTLA4, an RNA sequence transcribed from DNA encoding CTLA4 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding CTLA4. “CTLA4 mRNA” means an mRNA encoding a CTLA4 protein.

“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.

“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include an antisense compound and a target nucleic acid.

“IDO1 nucleic acid” means any nucleic acid encoding IDO1. For example, in certain embodiments, a IDO1 nucleic acid includes a DNA sequence encoding IDO1, an RNA sequence transcribed from DNA encoding IDO1 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding IDO1. “IDO1 mRNA” means an mRNA encoding an IDO1 protein.

“IDO2 nucleic acid” means any nucleic acid encoding IDO2. For example, in certain embodiments, a IDO2 nucleic acid includes a DNA sequence encoding IDO2, an RNA sequence transcribed from DNA encoding IDO2 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding IDO2. “IDO2 mRNA” means an mRNA encoding an IDO2 protein.

“Inhibiting PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA or protein expression” means reducing expression of PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA and/or protein levels in the presence of a gene silencing compound according to the invention as compared to expression of PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA and/or protein levels in the absence of a gene silencing compound according to the invention.

The term “kinase inhibitor” generally refers to molecules that antagonize or inhibit phosphorylation-dependent cell signaling and/or growth pathways in a cell. Kinase inhibitors may be naturally occurring or synthetic and include small molecules that have the potential to be administered as oral therapeutics. Kinase inhibitors have the ability to rapidly and specifically inhibit the activation of the target kinase molecules. Protein kinases are attractive drug targets, in part because they regulate a wide variety of signaling and growth pathways and include many different proteins. As such, they have great potential in the treatment of diseases involving kinase signaling, including cancer, cardiovascular disease, inflammatory disorders, diabetes, macular degeneration and neurological disorders. A non-limiting example of a kinase inhibitor is sorafenib.

“LAG3 nucleic acid” means any nucleic acid encoding LAG3. For example, in certain embodiments, a LAG3 nucleic acid includes a DNA sequence encoding LAG3, an RNA sequence transcribed from DNA encoding LAG3 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding LAG3. “LAG3 mRNA” means an mRNA encoding a LAG3 protein.

The term “linear synthesis” generally refers to a synthesis that starts at one end of an oligonucleotide and progresses linearly to the other end. Linear synthesis permits incorporation of either identical or non-identical (in terms of length, base composition and/or chemical modifications incorporated) monomeric units into an oligonucleotide.

The term “mammal” is expressly intended to include warm blooded, vertebrate animals, including, without limitation, humans, non-human primates, rats, mice, cats, dogs, horses, cattle, cows, pigs, sheep and rabbits.

The term “nucleoside” generally refers to compounds consisting of a sugar, usually ribose, deoxyribose, pentose, arabinose or hexose, and a purine or pyrimidine base.

The term “nucleotide” generally refers to a nucleoside comprising a phosphorous-containing group attached to the sugar.

The term “modified nucleoside” or “nucleotide derivative” generally is a nucleoside that includes a modified heterocyclic base, a modified sugar moiety, or any combination thereof. In some embodiments, the modified nucleoside or nucleotide derivative is a non-natural pyrimidine or purine nucleoside, as herein described. For purposes of the invention, a modified nucleoside or nucleotide derivative, a pyrimidine or purine analog or non-naturally occurring pyrimidine or purine can be used interchangeably and refers to a nucleoside that includes a non-naturally occurring base and/or non-naturally occurring sugar moiety. For purposes of the invention, a base is considered to be non-natural if it is not guanine, cytosine, adenine, thymine or uracil and a sugar is considered to be non-natural if it is not β-ribo-furanoside or 2′-deoxyribo-furanoside.

The term “modified oligonucleotide” as used herein describes an oligonucleotide in which at least two of its nucleotides are covalently linked via a synthetic linkage, i.e., a linkage other than a phosphodiester linkage between the 5′ end of one nucleotide and the 3′ end of another nucleotide in which the 5′ nucleotide phosphate has been replaced with any number of chemical groups. The term “modified oligonucleotide” also encompasses 2′-O,4′-C-methylene-b-D-ribofuranosyl nucleic acids, arabinose nucleic acids, substituted arabinose nucleic acids, hexose nucleic acids, peptide nucleic acids, morpholino, and oligonucleotides having at least one nucleotide with a modified base and/or sugar, such as a 2′-O-substituted, a 5-methylcytosine and/or a 3′-O-substituted ribonucleotide.

The term “nucleic acid” encompasses a genomic region or an RNA molecule transcribed therefrom. In some embodiments, the nucleic acid is mRNA.

The term “linker” generally refers to any moiety that can be attached to an oligonucleotide by way of covalent or non-covalent bonding through a sugar, a base, or the backbone. The non-covalent linkage may be, without limitation, electrostatic interactions, hydrophobic interactions, π-stacking interactions, hydrogen bonding and combinations thereof. Non-limiting examples of such non-covalent linkage includes Watson-Crick base pairing,

Hoogsteen base pairing, and base stacking. The linker can be used to attach two or more nucleosides or can be attached to the 5′ and/or 3′ terminal nucleotide in the oligonucleotide. Such linker can be either a non-nucleotide linker or a nucleoside linker.

The term “non-nucleotide linker” generally refers to a chemical moiety, other than a linkage directly between two nucleotides that can be attached to an oligonucleotide by way of covalent or non-covalent bonding. Preferably such non-nucleotide linker is from about 2 angstroms to about 200 angstroms in length, and may be either in a cis or trans orientation.

The term “internucleotide linkage” generally refer to a chemical linkage to join two nucleosides through their sugars (e.g. 3′-3′, 2′-3′, 2′-5′, 3′-5′, 5′-5′) consisting of a phosphorous atom and a charged, or neutral group (e.g., phosphodiester, phosphorothioate, phosphorodithioate or methylphosphonate) between adjacent nucleosides.

The term “oligonucleotide” refers to a polynucleoside formed from a plurality of linked nucleoside units, which may include, for example, deoxyribonucleotides or ribonucleotides, synthetic or natural nucleotides, phosphodiester or modified linkages, natural bases or modified bases natural sugars or modified sugars, or combinations of these components. The nucleoside units may be part of viruses, bacteria, cell debris or oligonucleotide-based compositions (for example, siRNA and microRNA). Such oligonucleotides can also be obtained from existing nucleic acid sources, including genomic or cDNA, but are preferably produced by synthetic methods. In certain embodiments each nucleoside unit includes a heterocyclic base and a pentofuranosyl, trehalose, arabinose, 2′-deoxy-2′-substituted nucleoside, 2′-deoxy-2′-substituted arabinose, 2′-O-substituted arabinose or hexose sugar group. The nucleoside residues can be coupled to each other by any of the numerous known internucleoside linkages. Such internucleoside linkages include, without limitation, phosphodiester, phosphorothioate, phosphorodithioate, methylphosphonate, alkylphosphonate, alkylphosphonothioate, phosphotriester, phosphoramidate, siloxane, carbonate, carboalkoxy, acetamidate, carbamate, morpholino, borano, thioether, bridged phosphoramidate, bridged methylene phosphonate, bridged phosphorothioate, and sulfone internucleoside linkages. The term “oligonucleotide” also encompasses polynucleosides having one or more stereospecific internucleoside linkage (e.g., (R_(P))- or (S_(P))-phosphorothioate, alkylphosphonate, or phosphotriester linkages). As used herein, the terms “oligonucleotide” and “dinucleotide” are expressly intended to include polynucleosides and dinucleosides having any such internucleoside linkage, whether or not the linkage comprises a phosphate group. In certain exemplary embodiments, these internucleoside linkages may be phosphodiester, phosphorothioate or phosphorodithioate linkages, or combinations thereof. In exemplary embodiments, the nucleotides of the synthetic oligonucleotides are linked by at least one phosphorothioate internucleotide linkage. The phosphorothioate linkages may be mixed Rp and Sp enantiomers, or they may be stereoregular or substantially stereoregular in either Rp or Sp form (see Iyer et al. (1995) Tetrahedron Asymmetry 6:1051-1054). In certain embodiments, one or more of the oligonucleotides within the antisense compositions of the invention contain one or more 2′-O,4′-C-methylene-b-D-ribofuranosyl nucleic acids, wherein the ribose is modified with a bond between the 2′ and 4′ carbons, which fixes the ribose in the 3′-endo structural conformation.

“OX40 nucleic acid” means any nucleic acid encoding OX40. For example, in certain embodiments, a OX40 nucleic acid includes a DNA sequence encoding OX40, an RNA sequence transcribed from DNA encoding OX40 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding OX40. “OX40 mRNA” means an mRNA encoding an OX40 protein.

“OX40L nucleic acid” means any nucleic acid encoding OX40L. For example, in certain embodiments, a OX40L nucleic acid includes a DNA sequence encoding OX40L, an RNA sequence transcribed from DNA encoding OX40L (including genomic DNA comprising introns and exons), and an mRNA sequence encoding OX40L. “OX40L mRNA” means an mRNA encoding an OX40L protein.

“PD1 nucleic acid” means any nucleic acid encoding PD1. For example, in certain embodiments, a PD1 nucleic acid includes a DNA sequence encoding PD1, an RNA sequence transcribed from DNA encoding PD1 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding PD1. “PD1 mRNA” means an mRNA encoding a PD1 protein.

“PDL1 nucleic acid” means any nucleic acid encoding PDL1. For example, in certain embodiments, a PDL1 nucleic acid includes a DNA sequence encoding PDL1, an RNA sequence transcribed from DNA encoding PDL1 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding PDL1. “PDL1 mRNA” means an mRNA encoding a PDL1 protein.

The term “peptide” generally refers to oligomers or polymers of amino acids that are of sufficient length and composition to affect a biological response, for example, antibody production or cytokine activity whether or not the peptide is a hapten. The term “peptide” may include modified amino acids (whether or not naturally or non-naturally occurring), where such modifications include, but are not limited to, phosphorylation, glycosylation, pegylation, lipidization, and methylation.

The term “pharmaceutically acceptable” means a non-toxic material that does not interfere with the effectiveness of a compound according to the invention or the biological activity of a compound according to the invention.

The term “physiologically acceptable” refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. Preferably, the biological system is a living organism, such as a mammal, particularly a human.

The term “prophylactically effective amount” generally refers to an amount sufficient to prevent or reduce the development of an undesired biological effect.

“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound.

“Single-stranded oligonucleotide” means an oligonucleotide which is not hybridized to a complementary strand.

“Specifically hybridizable” refers to a gene silencing compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments.

“Targeting” or “targeted” means the process of design and selection of a gene silencing compound that will specifically hybridize to a target nucleic acid and induce a desired effect.

“Target nucleic acid,” “target RNA,” “target mRNA,” and “target RNA transcript” all refer to a nucleic acid capable of being targeted by gene silencing compounds.

“Target segment” means the sequence of nucleotides of a target nucleic acid to which a gene silencing compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.

The term “therapeutically effective amount” or “pharmaceutically effective amount” generally refers to an amount sufficient to affect a desired biological effect, such as a beneficial result, including, without limitation, prevention, diminution, amelioration or elimination of signs or symptoms of a disease or disorder. Thus, the total amount of each active component of the pharmaceutical composition or method is sufficient to show a meaningful patient benefit, for example, but not limited to, healing of chronic conditions characterized by immune stimulation. Thus, a “pharmaceutically effective amount” will depend upon the context in which it is being administered. A pharmaceutically effective amount may be administered in one or more prophylactic or therapeutic administrations. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

“TIM3 nucleic acid” means any nucleic acid encoding TIM3. For example, in certain embodiments, a TIM3 nucleic acid includes a DNA sequence encoding TIM3, an RNA sequence transcribed from DNA encoding TIM3 (including genomic DNA comprising introns and exons), and an mRNA sequence encoding TIM3. “TIM3 mRNA” means an mRNA encoding a TIM3 protein.

The term “treatment” generally refers to an approach intended to obtain a beneficial or desired result, which may include alleviation of symptoms, or delaying or ameliorating a disease progression.

The term “gene expression” generally refers to process by which information from a gene is used in the synthesis of a functional gene product, which may be a protein. The process may involve transcription, RNA splicing, translation, and post-translational modification of a protein, and may include mRNA, preRNA, ribosomal RNA, and other templates for protein synthesis.

In certain embodiments provided are methods, compounds, and compositions for inhibiting PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L mRNA or protein expression. In certain embodiments the compounds are antisense oligonucleotides, double stranded or single-stranded siRNA compounds, or gene silencing compounds.

As used herein, gene silencing compounds according to the invention comprise two or more single-stranded antisense oligonucleotides linked at their 5′ ends, wherein the compounds have two or more accessible 3′ ends. The general structure of the oligonucleotide-based compounds of the invention may be described by the following formula I:

3′-Nn . . . N1N2N3N4-5′-L-5′-N8N7N6N5 . . . Nm-3′  (Formula I),

wherein L is a nucleotide linker or non-nucleotide linker; N1-N8, at each occurrence, is independently a nucleotide or nucleotide derivative; Nm and Nn, at each occurrence, are independently a nucleotide or nucleotide derivative; and wherein m and n are independently numbers from 0 to about 40.

The linkage at the 5′ ends of the component oligonucleotides is independent of the other oligonucleotide linkages and may be directly via 5′, 3′ or 2′ hydroxyl groups, or indirectly, via a non-nucleotide linker or a nucleoside, utilizing either the 2′ or 3′ hydroxyl positions of the nucleoside. Linkages may also utilize a functionalized sugar or nucleobase of a 5′ terminal nucleotide.

In certain embodiments provided are gene silencing compounds targeted to a mouse or human PD1 nucleic acid. In certain embodiments, the mouse PD1 nucleic acid is the sequence set forth in GENBANK Accession No. NM_008798 (incorporated herein as SEQ ID NO: 387) or the human PD1 nucleic acid is the sequence set forth in GENBANK Accession No. NM_005018 (incorporated herein as SEQ ID NO: 388).

In certain embodiments provided are gene silencing compounds targeted to a mouse or human PDL1 nucleic acid. In certain embodiments, the mouse PDL1 nucleic acid is the sequence set forth in GENBANK Accession No. NM_021893 (incorporated herein as SEQ ID NO: 389) or the human PDL1 nucleic acid is the sequence set forth in GENBANK Accession No. NM_014143 (incorporated herein as SEQ ID NO: 390).

In certain embodiments provided are gene silencing compounds targeted to a mouse or human IDO1 nucleic acid. In certain embodiments, the mouse IDO1 nucleic acid is the sequence set forth in GENBANK Accession No. NM_008324 (incorporated herein as SEQ ID NO: 391) or the human IDO1 nucleic acid is the sequence set forth in GENBANK Accession No. NM_002164 (incorporated herein as SEQ ID NO: 392).

In certain embodiments provided are gene silencing compounds targeted to a mouse or human LAG3 nucleic acid. In certain embodiments, the mouse LAG3 nucleic acid is the sequence set forth in GENBANK Accession No. NM_008479 (incorporated herein as SEQ ID NO: 393) or the human LAG3 nucleic acid is the sequence set forth in GENBANK Accession No. NM_002286 (incorporated herein as SEQ ID NO: 394).

In certain embodiments provided are gene silencing compounds targeted to a mouse or human TIM3 nucleic acid. In certain embodiments, the mouse TIM3 nucleic acid is the sequence set forth in GENBANK Accession No. NM_134250 (incorporated herein as SEQ ID NO: 395) or the human TIM3 nucleic acid is the sequence set forth in GENBANK Accession No. NM_032782 (incorporated herein as SEQ ID NO: 396).

In certain embodiments provided are gene silencing compounds targeted to a mouse or human CTLA4 nucleic acid. In certain embodiments, the mouse CTLA4 nucleic acid is the sequence set forth in GENBANK Accession No. NM_009843 (incorporated herein as SEQ ID NO: 397) or the human CTLA4 nucleic acid is the sequence set forth in GENBANK Accession No. NM_005214 (incorporated herein as SEQ ID NO: 398).

In certain embodiments provided are gene silencing compounds targeted to a mouse or human IDO2 nucleic acid. In certain embodiments, the mouse IDO2 nucleic acid is the sequence set forth in GENBANK Accession No. NM_145949 (incorporated herein as SEQ ID NO: 399) or the human IDO2 nucleic acid is the sequence set forth in GENBANK Accession No. NM_194294 (incorporated herein as SEQ ID NO: 400).

In certain embodiments provided are gene silencing compounds targeted to a mouse or human CEACAM1 nucleic acid. In certain embodiments, the mouse CEACAM1 nucleic acid is the sequence set forth in GENBANK Accession No. NM_001039187 (incorporated herein as SEQ ID NO: 401) or the human CEACAM1 nucleic acid is the sequence set forth in GENBANK Accession No. NM_001205344 (incorporated herein as SEQ ID NO: 402).

In certain embodiments provided are gene silencing compounds targeted to a mouse or human OX40 nucleic acid. In certain embodiments, the mouse OX40 nucleic acid is the sequence set forth in GENBANK Accession No. NM_011659 (incorporated herein as SEQ ID NO: 403) or the human OX40 nucleic acid is the sequence set forth in GENBANK Accession No. NM_003327 (incorporated herein as SEQ ID NO: 404).

In certain embodiments provided are gene silencing compounds targeted to a mouse or human OX40L nucleic acid. In certain embodiments, the mouse OX40L nucleic acid is the sequence set forth in GENBANK Accession No. NM_009452 (incorporated herein as SEQ ID NO: 405) or the human OX40L nucleic acid is the sequence set forth in GENBANK Accession No. NM_003326 (incorporated herein as SEQ ID NO: 406).

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, or SEQ ID NO: 406. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, or SEQ ID NO: 406. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, or SEQ ID NO: 406. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, or SEQ ID NO: 406.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401, SEQ ID NO: 403, or SEQ ID NO: 405. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401, SEQ ID NO: 403, or SEQ ID NO: 405. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401, SEQ ID NO: 403, or SEQ ID NO: 405. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401, SEQ ID NO: 403, or SEQ ID NO: 405.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 392, SEQ ID NO: 394, SEQ ID NO: 396, SEQ ID NO: 398, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, or SEQ ID NO: 406. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 392, SEQ ID NO: 394, SEQ ID NO: 396, SEQ ID NO: 398, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, or SEQ ID NO: 406. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 392, SEQ ID NO: 394, SEQ ID NO: 396, SEQ ID NO: 398, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, or SEQ ID NO: 406. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 392, SEQ ID NO: 394, SEQ ID NO: 396, SEQ ID NO: 398, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, or SEQ ID NO: 406.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 388. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 388. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 388. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 388.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 389. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 389. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 389. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 389.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 390. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 390. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 390. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 390.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 391. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 391. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 391. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 391.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 392. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 392. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 392. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 392.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 393. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 393. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 393. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 393.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 394. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 394. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 394. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 394.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 395. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 395. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 395. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 395.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 396. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 396. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 396. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 396.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 397. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 397. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 397. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 397.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 398. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 398. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 398. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 398.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 399. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 399. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 399. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 399.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 400. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 400. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 400. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 400.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 401. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 401. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 401. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 401.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 402. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 402. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 402. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 402.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 403. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 403. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 403. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 403.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 404. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 404. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 404. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 404.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 405. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 405. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 405. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 405.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides each, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 406. Certain embodiments provide compounds comprising two oligonucleotides each, independently, consisting of 15 to 25 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 406. Certain embodiments provide compounds comprising a modified oligonucleotide consisting of 18 to 21 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 406. In certain embodiments, the two oligonucleotide of the gene silencing compound each, independently, comprise at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 406.

In certain embodiments, the nucleobase sequence of the oligonucleotides of the gene silencing compound are, independently, at least 90% complementary over its entire length to a nucleobase sequence of SEQ ID NO: 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, or 406. In certain embodiments, the nucleobase sequence of the oligonucleotides of the gene silencing compound are, independently, at least 95% complementary over its entire length to a nucleobase sequence of SEQ ID NO: 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, or 406. In certain embodiments, the oligonucleotides of the gene silencing compound are at least 99% complementary over its entire length to SEQ ID NO: 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, or 406. In certain embodiments, the nucleobase sequence of the oligonucleotides of the gene silencing compound are 100% complementary over its entire length to a nucleobase sequence of SEQ ID NO: 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, or 406.

In certain embodiments, the oligonucleotides of the gene silencing compound are, independently, 12 to 30 nucleotides in length. In other words, the oligonucleotides are from 12 to 30 linked nucleobases. In other embodiments, the oligonucleotides, independently, consist of 15 to 28, 18 to 24, 19 to 22, or 20 linked nucleobases. In certain such embodiments, the oligonucleotides, independently, consist of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 linked nucleobases in length, or a range defined by any two of the above values.

In certain embodiments, a target region is a structurally defined region of the target nucleic acid. For example, a target region may encompass a 3′ UTR, a 5′ UTR, an exon, an intron, an exon/intron junction, a coding region, a translation initiation region, translation termination region, or other defined nucleic acid region. The structurally defined regions for PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L can be obtained by accession number from sequence databases such as NCB' and such information is incorporated herein by reference. In certain embodiments, a target region may encompass the sequence from a 5′ target site of one target segment within the target region to a 3′ target site of another target segment within the same target region.

Certain embodiments provide a composition comprising a 3GA compound as described herein, or a salt thereof, and a pharmaceutically acceptable carrier or diluent. Certain embodiments provide a composition comprising two or more 3GA compounds as described herein, or a salt thereof, and a pharmaceutically acceptable carrier or diluent. The two or more 3GA compounds can inhibit the mRNA or protein expression of the same target or can inhibit the mRNA or protein expression of different targets.

In certain embodiments, the 3GA compounds according to the invention comprise two identical or different sequences linked at their 5′-5′ ends via a phosphodiester, phosphorothioate or non-nucleoside linker. 3GA compounds according to the invention that comprise identical sequences are able to bind to a specific mRNA via Watson-Crick hydrogen bonding interactions and inhibit mRNA and protein expression. Gene silencing compounds according to the invention that comprise different sequences are able to bind to two or more different regions of one or more mRNA target and inhibit mRNA and protein expression. Such compounds are comprised of heteronucleotide sequences complementary to target mRNA and form stable duplex structures through Watson-Crick hydrogen bonding.

In certain embodiments, gene silencing compounds according to the invention are useful in treating and/or preventing diseases wherein inhibiting PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L expression would be beneficial. Gene silencing compounds according to the invention include, but are not limited to, antisense oligonucleotides comprising naturally occurring nucleotides, modified nucleotides, modified oligonucleotides and/or backbone modified oligonucleotides.

The oligonucleotides of the 3GA compounds are linked through their 5′-ends to allow the presence of two or more accessible 3′-ends. In certain embodiments, the oligonucleotides are linked through one or more of the non-nucleotide linkers listed in Table 1. In certain embodiments, a single linker listed in Table 1 is used to link the oligonucleotides of the gene silencing compounds. In certain embodiments, the linker is small molecule linker such as glycerol or a glycerol homolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and p independently are integers from 1 to about 6, from 1 to about 4 or from 1 to about 3. In some other embodiments, the small molecule linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH, wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 to about 6 or from 2 to about 4. Representative non-nucleotide linkers are set forth in Table 1.

TABLE 1 Representative Non-Nucleotide Linkers

Glycerol (1,2,3-Propanetriol)

1,2,4-Butanetriol

2-(hydroxymethyl)-1,3-propanediol

2-(hydroxymethyl)1,4-butanediol

1,3,5-Pentanetriol

1,1,1-Tris(hydroxymethyl)ethane

1,1,1-Tris(hydroxymethyl)nitromethane

1,1,1-Tris(hydroxymethyl)propane

1,2,6-Hexanetriol

3-Methyl-1,3,5-pentanetriol

1,2,3-Heptanetriol

2-Amino-2-(hydroxymethyl)-1,3-propanediol

N-[Tris(hydroxymethyl)methyl]acrylamide

cis-1,3,5-Cyclohexanetriol

cis-1,3,5-Tri(hydroxymethyl)cyclohexane

1,3,5-Trihydroxyl-benzene

3,5,-Di(hydroxymethyl)phenol

1,3,5,-Tri(hydroxymethyl)benzene

1,3-Di(hydroxyethoxy)-2-hydroxyl-propane

1,3-Di(hydroxypropoxy)-2-hydroxyl-propane

2-Deoxy-D-ribose

1,2,4,-Trihydroxyl-benzene

D-Galactoal

1,6-anhydro-β-D-Glucose

1,3,5-Tris(2-hydroxyethyl)-Cyanuric acid

Gallic acid

3,5,7-Trihydroxyflavone

4,6-Nitropyrogallol

Ethylene glycol

1,3-Propanediol

1,2-Propanediol

1,4-Butanediol

1,3-Butanediol

2,3-Butanediol

1,4-Butanediol

1,5-Pentanediol

2,4-Pentanediol

1,6-Hexanediol

1,2-Hexanediol

1,5-Hexanediol

2,5-Hexanediol

1,7-Heptanediol

1,8-Octanediol

1,2-Octanediol

1,9-Nonanediol

1,12-Dodecanediol

Triethylene glycol

Tetraethylene glycol

Hexaethylene glycol

2-(1-Aminopropyl)-1,3-propanediol

1,2-Dideoxyribose

In some embodiments, the small molecule linker is glycerol or a glycerol homolog of the formula HO—(CH₂)_(o)—CH(OH)—(CH₂)_(p)—OH, wherein o and p independently are integers from 1 to about 6, from 1 to about 4 or from 1 to about 3. In some other embodiments, the small molecule linker is a derivative of 1,3-diamino-2-hydroxypropane. Some such derivatives have the formula HO—(CH₂)_(m)—C(O)NH—CH₂—CH(OH)—CH₂—NHC(O)—(CH₂)_(m)—OH, wherein m is an integer from 0 to about 10, from 0 to about 6, from 2 to about 6 or from 2 to about 4.

In certain embodiments, the two or more oligonucleotides of the gene silencing compounds of the invention can be linked as shown in Table 2.

TABLE 2 Oligoribonucleotide Formulas II-V Formula II

Formula III

Formula IV

Formula V

In certain embodiments of Formulas II and/or V, L is a linker or a nucleotide linkage and Domain A and/or Domain B are antisense oligonucleotides that are designed to selectively hybridize to the same target RNA sequence or different target RNA sequences.

In certain embodiments of Formulas II, III, IV or V, L is a linker and Domain A and/or Domain B and/or Domain C and/or Domain D are antisense oligonucleotides that are designed to selectively hybridize to the same target RNA sequence or different target RNA sequences. For example, in one embodiment, Domain A and/or Domain B and/or Domain C of Formulas II and/or III are antisense oligonucleotides that are designed to selectively hybridize to the same target RNA sequence. In this embodiment, Domain A and/or Domain B and/or Domain C can be designed to hybridize to the same region on the target RNA sequence or to different regions of the same target RNA sequence.

In a further embodiment of this aspect of the invention, Domain A, Domain B, Domain C, and Domain D are independently RNA or DNA-based oligonucleotides. In certain aspects of this embodiment, the oligonucleotides comprise mixed backbone oligonucleotides.

In another embodiment, one or more of Domain A and/or Domain B and/or Domain C and/or Domain D is an antisense oligonucleotide that is designed to selectively hybridize to one target RNA sequence and one or more of the remaining Domain A and/or Domain B and/or Domain C and/or Domain D is an antisense oligonucleotide that is designed to selectively hybridized to a different target RNA sequence.

In another embodiment, one or more of Domain A and/or Domain B and/or Domain C and/or Domain D is an RNA-based oligonucleotide hybridized to a complimentary RNA-based oligonucleotide such that the domain comprises an siRNA molecule.

These gene silencing compounds of the invention can be prepared by the art recognized methods such as phosphoramidate or H-phosphonate chemistry which can be carried out manually or by an automated synthesizer. The synthetic antisense oligonucleotides of the invention may also be modified in a number of ways without compromising their ability to hybridize to mRNA. Such modifications may include at least one internucleotide linkage of the oligonucleotide being an alkylphosphonate, phosphorothioate, phosphorodithioate, methylphosphonate, phosphate ester, alkylphosphonothioate, phosphoramidate, carbamate, carbonate, phosphate hydroxyl, acetamidate or carboxymethyl ester or a combination of these and other internucleotide linkages between the 5′ end of one nucleotide and the 3′ end of another nucleotide in which the 5′ nucleotide phosphodiester linkage has been replaced with any number of chemical groups.

The synthetic antisense oligonucleotides of the invention may comprise combinations of internucleotide linkages. For example, U.S. Pat. No. 5,149,797 describes traditional chimeric oligonucleotides having a phosphorothioate core region interposed between methylphosphonate or phosphoramidate flanking regions. Additionally, U.S. Pat. No. 5,652,356 discloses “inverted” chimeric oligonucleotides comprising one or more nonionic oligonucleotide region (e.g. alkylphosphonate and/or phosphoramidate and/or phosphotriester internucleoside linkage) flanked by one or more region of oligonucleotide phosphorothioate. Various synthetic antisense oligonucleotides with modified internucleotide linkages can be prepared according to standard methods. In certain embodiments, the phosphorothioate linkages may be mixed Rp and Sp enantiomers, or they may be made stereoregular or substantially stereoregular in either Rp or Sp form.

Other modifications of gene silencing compounds of the invention include those that are internal or at the end(s) of the oligonucleotide molecule and include additions to the molecule of the internucleoside phosphate linkages, such as cholesterol, cholesteryl, or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose, deoxyribose and phosphate modifications which cleave, or crosslink to the opposite chains or to associated enzymes or other proteins which bind to the genome. Examples of such modified oligonucleotides include oligonucleotides with a modified base and/or sugar such as 2′-O,4′-C-methylene-b-D-ribofuranosyl, or arabinose instead of ribose, or a 3′, 5′-substituted oligonucleotide having a sugar which, at both its 3′ and 5′ positions, is attached to a chemical group other than a hydroxyl group (at its 3′ position) and other than a phosphate group (at its 5′ position).

Other examples of modifications to sugars of the oligonucleotide-based compounds of the invention include modifications to the 2′ position of the ribose moiety which include but are not limited to 2′-O-substituted with an -O-alkyl group containing 1-6 saturated or unsaturated carbon atoms, or with an -O-aryl, or -O-allyl group having 2-6 carbon atoms wherein such -O-alkyl, -0-aryl or -O-allyl group may be unsubstituted or may be substituted, for example with halo, hydroxyl, trifluoromethyl, cyano, nitro, acyl, acyloxy, alkoxy, carboxy, carbalkoxyl or amino groups. None of these substitutions are intended to exclude the presence of other residues having native 2′-hydroxyl group in the case of ribose or 2′ H- in the case of deoxyribose.

The gene silencing compounds according to the invention can comprise one or more ribonucleotides. For example, U.S. Pat No. 5,652,355 discloses traditional hybrid oligonucleotides having regions of 2′-O-substituted ribonucleotides flanking a DNA core region. U.S. Pat. No. 5,652,356 discloses an “inverted” hybrid oligonucleotide that includes an oligonucleotide comprising a 2′-O-substituted (or 2′ OH, unsubstituted) RNA region which is in between two oligodeoxyribonucleotide regions, a structure that “inverted relative to the “traditional” hybrid oligonucleotides. Non-limiting examples of particularly useful oligonucleotides of the invention have 2′-O-alkylated ribonucleotides at their 3′, 5′, or 3′ and 5′ termini, with at least four, and in some exemplary embodiments five, contiguous nucleotides being so modified. Non-limiting examples of 2′-O-alkylated groups include 2′-O-methyl, 2′-O-ethyl, 2′-O-propyl, 2′-O-butyls and 2′-O-methoxy-ethyl.

The oligonucleotide-based compounds of the invention may conveniently be synthesized using an automated synthesizer and phosphoramidite approach further described in Example 1. In some embodiments, the oligonucleotide-based compounds of the invention are synthesized by a linear synthesis approach.

An alternative mode of synthesis is “parallel synthesis”, in which synthesis proceeds outward from a central linker moiety. A solid support attached linker can be used for parallel synthesis, as is described in U.S. Pat. No. 5,912,332. Alternatively, a universal solid support (such as phosphate attached controlled pore glass) support can be used.

Parallel synthesis of the oligonucleotide-based compounds of the invention has several advantages over linear synthesis: (1) parallel synthesis permits the incorporation of identical monomeric units; (2) unlike in linear synthesis, both (or all) the monomeric units are synthesized at the same time, thereby the number of synthetic steps and the time required for the synthesis is the same as that of a monomeric unit; and (3) the reduction in synthetic steps improves purity and yield of the final immune modulatory oligoribonucleotide product.

At the end of the synthesis by either linear synthesis or parallel synthesis protocols, the oligonucleotide-based compounds of the invention may conveniently be deprotected with concentrated ammonia solution or as recommended by the phosphoramidite supplier, if a modified nucleoside is incorporated. The product oligonucleotide-based compounds is preferably purified by reversed phase HPLC, detritylated, desalted and dialyzed.

In certain embodiments, the oligonucleotides of the gene silencing compound according to the invention are selected from the non-limiting list of the oligonucleotides shown in Table 3 below. The oligonucleotides shown in Table 3 have phosphorothioate (PS) linkages, but may also include phosphodiester linkages. Those skilled in the art will recognize, however, that other linkages, based on phosphodiester or non-phosphodiester moieties may be included.

TABLE 3 Oligo #/ SEQ ID Target NO: Target Species Site Sequence 5′ → 3′ 1 PD1 Mouse 58 GCCGGACCCACATGCCCAG 2 PD1 Mouse 65 GGTACCTGCCGGACCCACA 3 PD1 Mouse 115 GCCACCCTGATTGCCAGCT 4 PD1 Mouse 198 GGTGGCATTTGCTCCCTCT 5 PD1 Mouse 755 GGTGTCTTCTCTCGTCCCT 6 PD1 Mouse 848 GCTGAGCCCCTACGTCCCA 7 PD1 Mouse 1161 CCCCAGCTCTGCACCTTGT 8 PD1 Mouse 1589 CTAGCTCTGCTGGTTCCCT 9 PD1 Human 69 GCGCCTGTGGGATCTGCAT 10 PD1 Human 108 GCCAGCCCAGTTGTAGCAC 11 PD1 Human 285 GCTTGTCCGTCTGGTTGCT 12 PD1 Human 495 CCCTTCTCTCTGTCACCCT 13 PD1 Human 496 GCCCTTCTCTCTGTCACCC 14 PD1 Human 497 TGCCCTTCTCTCTGTCACC 15 PD1 Human 616 GCCAGGACCCAGACTAGCA 16 PD1 Human 620 GACGGCCAGGACCCAGACT 17 PD1 Human 895 CCATCCTCAGGCCTCAGTG 18 PD1 Human 897 GTCCATCCTCAGGCCTCAG 19 PD1 Human 899 GTGTCCATCCTCAGGCCTC 20 PD1 Human 901 CAGTGTCCATCCTCAGGCC 21 PD1 Human 1003 GCACCCTGCCTGCTTCTCC 22 PD1 Human 1005 CTGCACCCTGCCTGCTTCT 23 PD1 Human 1137 GTGACACCTGCTGCCTGGG 24 PD1 Human 1161 ATCTGGCCCTCCCTGTAGG 25 PD1 Human 1163 GCATCTGGCCCTCCCTGTA 26 PD1 Human 1165 CTGCATCTGGCCCTCCCTG 27 PD1 Human 1167 GACTGCATCTGGCCCTCCC 28 PD1 Human 1169 GTGACTGCATCTGGCCCTC 29 PD1 Human 1412 CTCCTGTGCCCAGTCTTGG 30 PD1 Human 1512 CCCACCACAGCCAGGAGCT 31 PD1 Human 1513 GCCCACCACAGCCAGGAGC 32 PD1 Human 1563 GCCTGAGGTGCTGCCTGGG 33 PD1 Human 1591 CTGCCTCAGCTTCCCTGCC 34 PD1 Human 1592 ACTGCCTCAGCTTCCCTGC 35 PD1 Human 1615 CCTCCAGCTCTGCCTGCCC 36 PD1 Human 1616 GCCTCCAGCTCTGCCTGCC 37 PD1 Human 1720 GCCCTCCTGACCTTGGGAC 38 PD1 Human 1722 CTGCCCTCCTGACCTTGGG 39 PD1 Human 1724 CCCTGCCCTCCTGACCTTG 40 PD1 Human 1894 CCTTCCCACCCAGGCCCTG 41 PD1 Human 1896 TACCTTCCCACCCAGGCCC 42 PD1 Human 1898 TGTACCTTCCCACCCAGGC 43 PD1 Human 1900 CCTGTACCTTCCCACCCAG 44 PD1 Human 1996 CTGGATGCTGGTGGCCCTG 45 PD1 Human 1997 CCTGGATGCTGGTGGCCCT 46 PD1 Human 2024 CCCAGCCACTCAGGTGCCT 47 PD1 Human 2032 TCCCTTGTCCCAGCCACTC 48 PD1 Human 2034 GATCCCTTGTCCCAGCCAC 49 PDL1 Mouse 219 CAAGCAGGTCCAGCTCCCG 50 PDL1 Mouse 316 CTCCCCCTGAAGTTGCTGT 51 PDL1 Mouse 436 TTGTAGTCCGCACCACCGT 52 PDL1 Mouse 1399 GGTGACCTCTGTGTTCCCT 53 PDL1 Mouse 2152 GCCTGCCTCTGCCTCCCTA 54 PDL1 Mouse 3311 GCCCAGCCTGTTCCTTCAG 55 PDL1 Human 571 GGTAGCCCTCAGCCTGACA 56 PDL1 Human 892 CCATCATTCTCCCTTTTCT 57 PDL1 Human 1075 ATTGCCTGCATCCCACGGG 58 PDL1 Human 1080 CCCACATTGCCTGCATCCC 59 PDL1 Human 1103 TTCAGTGCTTGGGCCTTTT 60 PDL1 Human 1163 GGCTCCCTGTTTGACTCCA 61 PDL1 Human 1182 GTATCAAGGTCTCCCTCCA 62 PDL1 Human 1230 TCCTTTCTCCCTGTCACAG 63 PDL1 Human 1296 ATTCTCAACCCGTCTTCCT 64 PDL1 Human 1855 TCTGTTTGCTTCCTCAGCT 65 PDL1 Human 1904 GGGTGGCAGTCTGAGGTCT 66 PDL1 Human 1911 GGACAGTGGGTGGCAGTCT 67 PDL1 Human 2142 TTCCCCTCGCATCATCCTT 68 PDL1 Human 2192 TCCCAGACCACATTGGCCT 69 PDL1 Human 2901 TGCACCCTGGAGAGCCCAT 70 PDL1 Human 3128 GCTGGTGGCATTCAAGGGT 71 PDL1 Human 3173 CGAAACCTCCAGGAAGCCT 72 PDL1 Human 3196 GATCTCCCAGGGCATCTGA 73 PDL1 Human 3397 GCCTTGCTCAGCCACAATT 74 PDL1 Human 3402 TATGTGCCTTGCTCAGCCA 75 IDO1 Mouse 138 CTAGCCACAAGGACCCAGG 76 IDO1 Mouse 264 ATGTACCCCAGGGCCAGGT 77 IDO1 Mouse 295 ATCCCCTCGGTTCCACACA 78 IDO1 Mouse 492 CCCTTGTCGCAGTCCCCAC 79 IDO1 Mouse 817 GAAGATGCTGCTCTGGCCT 80 IDO1 Mouse 1145 CAGTCCCTCTGCTTTCCAC 81 IDO1 Human 172 GCAGAGCAAAGCCCACTTC 82 IDO1 Human 184 CCTGTGGATTTGGCAGAGC 83 IDO1 Human 388 CTCCATGACCTTTGCCCCA 84 IDO1 Human 507 CTTTTTCTTCCAGTTTGCC 85 IDO1 Human 619 CAGCTGCTATTTCCACCAA 86 IDO1 Human 816 GTTGCCTTTCCAGCCAGAC 87 IDO1 Human 823 GCTGGGGGTTGCCTTTCCA 88 IDO1 Human 849 CCCTTCATACACCAGACCG 89 IDO1 Human 956 TGTCCTCCACCAGCAGTCT 90 IDO1 Human 1138 GCAGATGGTAGCTCCTCAG 91 IDO1 Human 1187 TCCTTTGGCTGCTGGCTTG 92 IDO1 Human 1239 GCCTCCAGTTCCTTTGGCT 93 IDO1 Human 1246 AATCAGTGCCTCCAGTTCC 94 IDO1 Human 1327 GTGCTCTTGTTGGGTTACA 95 IDO1 Human 1627 GCCTCGGCCTCCCAAAGTG 96 IDO1 Human 1745 TAGCTGGGACTACAGGTGC 97 IDO1 Human 1767 TCTCCTGCCTCAGCCTCCC 98 IDO1 Human 1774 ACGCCATTCTCCTGCCTCA 99 IDO1 Human 1792 GCTCCGCCTCCCAGGTTCA 100 IDO1 Human 1815 GGCACAATCTTGGCTCACT 101 LAG3 Mouse 25 GCTCCTCCAGACCCAGTCC 102 LAG3 Mouse 321 GGCCTCCCCAGCCCTCCAA 103 LAG3 Mouse 355 GGAGCAGGTCCTCCCTCAT 104 LAG3 Mouse 422 AGCTCTTTCCCAGGCCCTG 105 LAG3 Mouse 585 CCCCTGGTGAAGGTCAAGG 106 LAG3 Mouse 590 GGCATCCCCTGGTGAAGGT 107 LAG3 Mouse 601 GTCTAGGCGAGGGCATCCC 108 LAG3 Mouse 953 GGCACTCGGTTCTGGCCCT 109 LAG3 Mouse 1044 GACACAGCCCCAGGTCCCA 110 LAG3 Mouse 1108 GCTCCAGACCCAGAACCTT 111 LAG3 Mouse 1161 GGGCAGCTCCACCCTAGAA 112 LAG3 Mouse 1260 GCCACTCTTTCCAGCCACG 113 LAG3 Mouse 1295 GCCAGACCCACAGCCTCAA 114 LAG3 Mouse 1316 CAGGTGTAGGTCCCAGCCT 115 LAG3 Mouse 1349 GCATTGAGCTGCTGTCCCT 116 LAG3 Mouse 1524 GGCCTCCTGAATCTCCAGC 117 LAG3 Mouse 1573 GCCTCTGGCCCTCGTACAG 118 LAG3 Mouse 1819 CCAGCTCCTCTATCTTCCT 119 LAG3 Mouse 1918 CTGCCTCGGCTCCAGGTCA 120 LAG3 Mouse 1936 GCTGCTGAGACCTGCTGGC 121 LAG3 Mouse/ 1315 AGGTGTAGGTCCCAGCCTG Human 122 LAG3 Mouse/ 1822 GCTCCAGCTCCTCTATCTT Human 123 LAG3 Mouse/ 1062 GCCATCTCTGTAGGTGAGG Human 124 LAG3 Mouse/ 1356 GACAGTGGCATTGAGCTGC Human 125 LAG3 Human 3 TCTCTGGGCCTTCACCCCT 126 LAG3 Human 123 CTGGGCAGATCAGGCAGCC 127 LAG3 Human 167 GGGAGGGATGACCAGAGGC 128 LAG3 Human 229 GGGAGGTGGAGGAAGGGGT 129 LAG3 Human 346 CTGAGCCTCCCACATCTCT 130 LAG3 Human 395 GCTTCACTGGAGCCACCCA 131 LAG3 Human 494 GGCTGAGATCCTGGAGGGG 132 LAG3 Human 524 GCTGCCAAGTGACCCCTGC 133 LAG3 Human 648 GGACCCACGCTCAGCACCG 134 LAG3 Human 736 CCATAGCGAGAAGTCCCCG 135 LAG3 Human 834 TGGCCCAGGCGCAGACGGA 136 LAG3 Human 1034 CCATGGGGCTGACTTGGGG 137 LAG3 Human 1359 TTGAGCTGCTGTTCCTGCA 138 LAG3 Human 1433 GCAGCTTCCCCAGGGATCC 139 LAG3 Human 1499 GGGATGGGGTGTCCAGAGA 140 LAG3 Human 1554 TGGGAAAGGAGCTGGGCCT 141 LAG3 Human 1593 AGAAGCCTCTCCCCCTGGT 142 LAG3 Human 1636 GGCACCTGGGCTAGACAGC 143 LAG3 Human 1848 GGTTCTTGCTCCAGCTCCT 144 LAG3 Human 1940 GCTGAGATCTGCTGGCTGC 145 LAG3 Human/ 1972 GCTGCTGACAGGGAGTTTA Mouse 146 LAG3 Human/ 642 ACGCTCAGCACCGTGTAGC Mouse 147 LAG3 Human/ 1234 AGGAGGAGTCCACTTGGCA Mouse 148 LAG3 Human/ 1366 AGTGGCATTGAGCTGCTGT Mouse 149 TIM3 Mouse 222 AATCCCTTGCCCCAGCACA 150 TIM3 Mouse 319 GAGATCGCCCTTTAGCTGG 151 TIM3 Mouse 386 TGCAGCAGTAGGTCCCATG 152 TIM3 Mouse 462 GGAGTGACCTTGGCTGCTT 153 TIM3 Mouse 661 CCCAGCAGAGACTCCCACT 154 TIM3 Mouse 782 CATTTGCCAACCCTCCTGG 155 TIM3 Mouse 887 GCTGGCTGTTGACGTAGCA 156 TIM3 Mouse 1273 TTAGCCCTTTATTCCCCCT 157 TIM3 Mouse 1416 CCTCCTGCCTAAGGTTCCC 158 TIM3 Mouse 1425 ACTTATCACCCTCCTGCCT 159 TIM3 Mouse 1517 GAGCCTCATCTCCAGCCTC 160 TIM3 Mouse 1526 TCACTGTCCGAGCCTCATC 161 TIM3 Mouse 1668 CTGACTGCACGCAAGCCCC 162 TIM3 Mouse 1767 GAGCAGAGGACAACCCCCA 163 TIM3 Mouse 1953 CTGCTCTGCCATGCTCCCA 164 TIM3 Mouse 2138 GTCAGTTCCCCTTGAGCAC 165 TIM3 Mouse 2220 CTGCCTTCGTATGTCCCAG 166 TIM3 Mouse 2461 CACAGTTGCTCCCCAATGC 167 TIM3 Mouse 2570 AGCCAGGACCTCCACAGCT 168 TIM3 Mouse 2596 GTCTCCCTTCCATACCCAC 169 TIM3 Human 59 CTGCCAGGTCTACAGTCAC 170 TIM3 Human 281 CAGCAGCAGCAGCAGGACA 171 TIM3 Human 338 GGCATTCTGACCGACCTCC 172 TIM3 Human 457 TCCCTTTCATCAGTCCTGA 173 TIM3 Human 740 GAGGCTCCCCAGTGTCTGT 174 TIM3 Human 803 GGCCAATCTAGAGTCCCGT 175 TIM3 Human 1110 GTGAGGGTTGCTGCCTGCT 176 TIM3 Human 1235 GCAGTGGACAGAACCTCCA 177 TIM3 Human 1304 CAGTGCAGGTCCCAGTTCA 178 TIM3 Human 1442 GAGCTCCAGAGACCCCACG 179 TIM3 Human 1456 GCCCGAATTTCCTGGAGCT 180 TIM3 Human 1506 CAGCACCCAGTTTTCCCTA 181 TIM3 Human 1549 GCCCCTTTAGACTTTCTGT 182 TIM3 Human 1640 TGCCATTGCACTCCAGCCT 183 TIM3 Human 1716 ATCCCAGCCACTCAGGAGG 184 TIM3 Human 1725 ATGCCTGTAATCCCAGCCA 185 TIM3 Human 1863 GCTCACGCCTGTAATCCCA 186 TIM3 Human 1877 GGCTGGATGTGGTGGCTCA 187 TIM3 Human 2053 GCCACATCTCAGCCCTGCA 188 TIM3 Human 2246 GCCTTTGCCTTCTTTCCAC 189 CTLA4 Mouse 106 GGTCCTCAGGGAGCAGAGT 190 CTLA4 Mouse 191 AGGCCAAGTCCTAGAAGGC 191 CTLA4 Mouse 253 TGGGTCACCTGTATGGCTT 192 CTLA4 Mouse 344 AGTCACCCGGACCTCATCA 193 CTLA4 Mouse 416 GCCCACTGTATTCTTCTCT 194 CTLA4 Mouse 497 GTCAACAGCTCTCAGTCCT 195 CTLA4 Mouse 563 GTTGCCCATGCCCACAAAG 196 CTLA4 Mouse 567 TCCCGTTGCCCATGCCCAC 197 CTLA4 Mouse 647 CCCCAAGCTAACTGCGACA 198 CTLA4 Mouse 735 TCACATAGACCCCTGTTGT 199 CTLA4 Mouse 760 CATTCTGGCTCTGTTGGGG 200 CTLA4 Mouse 1084 CCTTGACCCCACACCATAA 201 CTLA4 Mouse 1135 CTCTTCCTTCACCCCCTTC 202 CTLA4 Mouse 1434 CTCCCCAGCCAAACCTCCC 203 CTLA4 Mouse 1436 AGCTCCCCAGCCAAACCTC 204 CTLA4 Mouse 1470 GACCTCGAGTCCAACCTGA 205 CTLA4 Mouse 1484 GCCAGTTGGTGCAGGACCT 206 CTLA4 Mouse 1542 ACTCCATCACCATCGGTTT 207 CTLA4 Mouse 1552 CCCAGTTTACACTCCATCA 208 CTLA4 Mouse 1794 TCCCATCCTACCATCTGCT 209 CTLA4 Human 129 GGGAGCGGTGTTCAGGTCT 210 CTLA4 Human 211 AGGAGAGTGCAGGGCCAGG 211 CTLA4 Human 346 CGGACCTCAGTGGCTTTGC 212 CTLA4 Human 504 CCATGGCCCTCAGTCCTTG 213 CTLA4 Human 574 CCGTTGCCTATGCCCAGGT 214 CTLA4 Human 953 GGGTTCCGCATCCAACTTT 215 CTLA4 Human 1007 CATCCCAGCTCTGTCTTTC 216 CTLA4 Human 1067 GCATCCCCATATTAATCCC 217 CTLA4 Human 1136 CTCCCTGCCTTTTCCTTCT 218 CTLA4 Human 1308 ACCTTTAGCATCACTGGCT 219 CTLA4 Human 1514 AGTGTCCTGAGCTCCTCCA 220 CTLA4 Human 1537 CCTTGTGTTCTACCTGGTG 221 CTLA4 Human 1570 CCTCATCCAGTTTCCAAGC 222 CTLA4 Human 1606 CTCAGCACAATTCCACGCA 223 CTLA4 Human 1632 AGCCCCAAAGCACATGTCA 224 CTLA4 Human 1747 ATACCTGTGGGTCTCCTGG 225 CTLA4 Human 1822 GCCTTCTTCTGTCCATGGC 226 CTLA4 Human 1844 GCACCCCATTCTGCCACCT 227 CTLA4 Human/ 744 TCACATAGACCCCTGTTGT Mouse 228 CTLA4 Human/ 1117 TTGGGCTGTGCCATTCCCT Mouse 229 IDO2 Mouse 49 TGCCCCAGAGGAATGCCCA 230 IDO2 Mouse 127 GTGGTATCTCCCCAAGGAC 231 IDO2 Mouse 279 CAGTCCAGGAGAGGCATCC 232 IDO2 Mouse 440 GGAGTCCCAAGTTCCTGGA 233 IDO2 Mouse 510 TCCAACGGTCCTTCTGGGT 234 IDO2 Mouse 639 GCCTCCATTCCCTGAACCA 235 IDO2 Mouse 801 GGATTGTCCTTCCACCCAG 236 IDO2 Mouse 873 GCTGCACTTCCTCCAGAGT 237 IDO2 Mouse 971 GCGGCATGTAGTCCCTCAT 238 IDO2 Mouse 1047 CCAGGACCAGAGGCCAGTA 239 IDO2 Mouse 1215 GTACCCCCAGTGCCCCTGT 240 IDO2 Mouse 1280 CACCAGGACACAGGAGGGC 241 IDO2 Mouse 1617 GCTCCCACGGGACCTGACT 242 IDO2 Mouse 1782 TGAGGAGGTCATGGCTGCA 243 IDO2 Mouse 1911 GGGACGAGGGAGGTAGGGA 244 IDO2 Mouse 2058 GTTTGAGGCCCATCAGACC 245 IDO2 Mouse 2345 GCTCAGTGGCTCATCCCTG 246 IDO2 Mouse 2638 GGCTGTCCCAGGTCACAGA 247 IDO2 Mouse 2748 GGTGACTTCCAGGTCTGCA 248 IDO2 Mouse 2756 CCCGTGCTGGTGACTTCCA 249 IDO2 Human 156 GGTGTCCATTGCCTTCTGT 250 IDO2 Human 214 GCCTGGTGGGTGAAGTGTC 251 IDO2 Human 222 TTGTGGTGGCCTGGTGGGT 252 IDO2 Human 284 ATTCGGTCTGTGGGGCTCC 253 IDO2 Human 561 CTCCTTCCTGCCAGACATA 254 IDO2 Human 633 GCCCCAAGTTCCTGGAGAC 255 IDO2 Human 713 CCCAATTTCCAGGAATCCG 256 IDO2 Human 722 CTCCAGGTTCCCAATTTCC 257 IDO2 Human 757 TGCAGGCTCTCTCCCCCAG 258 IDO2 Human 802 GGCACTGCTTCTTTCTCTA 259 IDO2 Human 1137 AGTCACCACTTTCCTTGCT 260 IDO2 Human 1207 GGTGCTGAGTGGATGTCTT 261 IDO2 Human 1253 CAGCAAGTGGTCCTGTCCA 262 IDO2 Human 1363 GGCTTCCCATGCTTTGCCT 263 IDO2 Human 1415 TCCACCTGTGCCCCTGTCT 264 IDO2 Human 1464 ACTCCAAGGTCTTATCCCT 265 IDO2 Human 1573 TGATCCCAGGCAGAACCCT 266 IDO2 Human 1593 GGGCTGAGATCCTTCCTGG 267 IDO2 Human 1745 TGGGGGTTCTGCATGAGGA 268 IDO2 Human 1752 ACTCCTCTGGGGGTTCTGC 269 IDO2 Human 1837 AGTAATGTATCCCCAGGCA 270 IDO2 Human 1945 AAGAGGGCTGGTCTGGGAC 271 CEACAM1 Mouse 291 GTAGTGTTTCCCTTGTACC 272 CEACAM1 Mouse 294 GCCGTAGTGTTTCCCTTGT 273 CEACAM1 Mouse 299 CTATAGCCGTAGTGTTTCC 274 CEACAM1 Mouse 1110 GTGAGGAACAGAATCCGGG 275 CEACAM1 Mouse 1526 TTCCTGCTTCTGGTTTGTT 276 CEACAM1 Mouse 1530 CCATTTCCTGCTTCTGGTT 277 CEACAM1 Mouse 1531 GCCATTTCCTGCTTCTGGT 278 CEACAM1 Mouse 2474 CCATGCTGGAACTCTGTCT 279 CEACAM1 Mouse 2485 CTGCACAGGCTCCATGCTG 280 CEACAM1 Mouse 2486 CCTGCACAGGCTCCATGCT 281 CEACAM1 Mouse 2500 CTGTGGGATTGAAACCTGC 282 CEACAM1 Mouse 2507 GGTGTTACTGTGGGATTGA 283 CEACAM1 Mouse 2513 GCAGAAGGTGTTACTGTGG 284 CEACAM1 Mouse 2533 GTCTGAGCAGGTGGGGTGC 285 CEACAM1 Mouse 2536 GCAGTCTGAGCAGGTGGGG 286 CEACAM1 Mouse 2568 TGTCCAGGTAGCCAGGCCT 287 CEACAM1 Mouse 2570 AATGTCCAGGTAGCCAGGC 288 CEACAM1 Human 103 GCCCTGTCTTCACCTGTGG 289 CEACAM1 Human 111 TCCTGCTGGCCCTGTCTTC 290 CEACAM1 Human 126 GTGCCCCATGGTGTCTCCT 291 CEACAM1 Human 1021 TGGCGTGGCAGGTATAGGA 292 CEACAM1 Human 1403 GCCCCAGGTGAGAGGCCAT 293 CEACAM1 Human 1440 AACCAGGGCCACTACTCCA 294 CEACAM1 Human 1463 GCCAGGGCTACTGCTATCA 295 CEACAM1 Human 1851 GGTTTCCTACAGACTCCCA 296 CEACAM1 Human 1908 GTTCTGGTCCCTCTTTCCC 297 CEACAM1 Human 2230 GGTGCTTAGACCCTGATCC 298 CEACAM1 Human 2396 CTGCCTTGAACAGAGCCCA 299 CEACAM1 Human 2414 AACCCCTCCCTCTCAGCAC 300 CEACAM1 Human 2436 GCTGGTTCCCTCCTGAAGC 301 CEACAM1 Human 2473 CCTTTCCCAAGTTCCTAGC 302 CEACAM1 Human 2489 GGGCAGCTCTCTGATTCCT 303 CEACAM1 Human 2700 GCTCCTGACCAAGGGACCT 304 CEACAM1 Human 2894 AGCAGAGGCCAAGGTTTCC 305 CEACAM1 Human 2924 CTCCCACTTCTCAAGGACC 306 CEACAM1 Human 3019 TCACAGCCCCATTTCCCCA 307 CEACAM1 Human 3323 GCACAGTCCGTGTCAGGGT 308 OX40 Mouse 20 GTATGCAGAGTCCCATGAT 309 OX40 Mouse 121 CCTTGCAGGGTGTGGCTAT 310 OX40 Mouse 161 CCTTGTCTGCTTTCTGCCT 311 OX40 Mouse 270 TGTGACCACTGGGGTAGGT 312 OX40 Mouse 495 GAGGTTGGGTGCCTGGTCT 313 OX40 Mouse 509 GCCGCTGTCCTGCCGAGGT 314 OX40 Mouse 544 GGAGGGCAGGGAACACAGT 315 OX40 Mouse 572 CTGGTTGTTGCCTGGAGAA 316 OX40 Mouse 593 ATTGGTCCAGGGCTTGCAG 317 OX40 Mouse 642 CCAAGCTGTCACTGGCTGG 318 OX40 Mouse 693 GGGTCTCCCAGAGCAGTGT 319 OX40 Mouse 845 AGTCAAGGGAGCCAGCAGG 320 OX40 Mouse 904 GGTTTGGGAGTGTTAGGCA 321 OX40 Mouse 941 CTCCTGGATCGGGGTCCTG 322 OX40 Mouse 1010 GCCCCATAAAATCCACTCC 323 OX40 Mouse 1021 GGGTTGTCCGTGCCCCATA 324 OX40 Mouse 1038 GGCAGGCATCAGGATATGG 325 OX40 Mouse 1069 GCCCAGCACCTAGAACGGT 326 OX40 Mouse 1080 GCCCAGAGCCAGCCCAGCA 327 OX40 Mouse 1126 TTAGGAGCACCACCAGGCA 328 OX40 Human 82 CCCAGGAGGAGCAGAGCCG 329 OX40 Human 192 TGCAGCGGCTCACCATCCC 330 OX40 Human 273 AGGGCTTGCACGGCTTGGA 331 OX40 Human 300 TCCCACTTCTGAGGTTACA 332 OX40 Human 312 GCTTCCGCTCACTCCCACT 333 OX40 Human 342 AGACTGTGTCCTGTGTGGC 334 OX40 Human 347 GCGGCAGACTGTGTCCTGT 335 OX40 Human 401 GGCACAGTCAACTCCAGGC 336 OX40 Human 462 AGTTGGTCCAGGGCTTGCA 337 OX40 Human 485 GGTGTGCTTCCCAGCCAAG 338 OX40 Human 746 CCGGAGCAGGTACAGGGCC 339 OX40 Human 762 GCAGCCTCTGGTCCCTCCG 340 OX40 Human 763 GGCAGCCTCTGGTCCCTCC 341 OX40 Human 823 TGCTCCTCTTGGATGGGGG 342 OX40 Human 865 GGCCCAGGTCAGATCTTGG 343 OX40 Human 967 GTTGGCCCAGGAGCGTGGC 344 OX40 Human 1036 GCAGGAGGTATGCATGGCA 345 OX40 Human 1058 GTTTTTATTGTGGTCCCGC 346 OX40 Human 1075 GACTCCCGTCTGCCAAGGT 347 OX40L Mouse 141 CCCTTCCCCTTCCATCTCT 348 OX40L Mouse 167 TCCAGATTCTCATCCAGGG 349 OX40L Mouse 182 GGCCTTGATCCGTTTTCCA 350 OX40L Mouse 218 ACCACCAGCCTTAGCGTCT 351 OX40L Mouse 226 TCCCAGAGACCACCAGCCT 352 OX40L Mouse 240 CCCTGCTCCCTTGATCCCA 353 OX40L Mouse 303 TGGAGGGTCCTTTGCCGGA 354 OX40L Mouse 399 GTTCTGCACCTCCATAGTT 355 OX40L Mouse 454 AGGAGCCCTTCAGGTAGAT 356 OX40L Mouse 565 CCAAAGAGGCCACCACAGT 357 OX40L Mouse 650 ACAATCAGCTCCCCATCAT 358 OX40L Mouse 753 CCTGTGTCCCGTCCACCCT 359 OX40L Mouse 817 AGGGTAGGCTCTGCATTCA 360 OX40L Mouse 895 GCAGGCTCAAGGCAATCCT 361 OX40L Mouse 1069 TGGACACCACCCTTTCCAT 362 OX40L Mouse 1157 CCCCCATGAGATGAGAGAC 363 OX40L Mouse 1173 AATCTTCTTTCCAAGCCCC 364 OX40L Mouse 1193 AGTCCTGCTTTCCACGGGG 365 OX40L Mouse 1298 GGTGGGTATCATAGTCCCT 366 OX40L Mouse 1439 CCTTCTTGGCCTTTATCCT 367 OX40L Human 494 GGGCTCCTCATCCTTCTGG 368 OX40L Human 712 GTTCATGCTGGTGCCTGGT 369 OX40L Human 814 GGGAGGGCCAGGATCTGCT 370 OX40L Human 1104 CCTTCACTCCTTGCTCCTC 371 OX40L Human 1120 GATTCATAACCCCACTCCT 372 OX40L Human 1139 GTTCATACCACCTTTGGCA 373 OX40L Human 1276 GGCTCTCTTCAAGTCCTGA 374 OX40L Human 1378 CACATCCCCAGACAGTTCT 375 OX40L Human 1383 AGCATCACATCCCCAGACA 376 OX40L Human 1492 GTCCAGTTCCCTGCTATCC 377 OX40L Human 1569 TGCTTTGCCTGTCTGTGGC 378 OX40L Human 1577 GCATGTGTTGCTTTGCCTG 379 OX40L Human 1828 ATTCCATTGAAGCCCTGGC 380 OX40L Human 2127 CAGCCCTCCACCTTTCTGG 381 OX40L Human 2367 GTCCACAGTAGGCCCTCCA 382 OX40L Human 2376 CAGTGCCTGGTCCACAGTA 383 OX40L Human 2387 AGTATTTAGCCCAGTGCCT 384 OX40L Human 2729 CCCAAAGCGAGTGAGCACC 385 OX40L Human 2754 ACATGGGAAGAGCAGGCCA 386 OX40L Human 2808 GGTGGAGTGAGGCTGGTGC

Compound names for the 3^(rd) generation antisense (3GA) compounds according to the invention are based on the target and oligonucleotide target site(s) as depicted Table 3. For example, “3GA 384” comprises two copies of Oligo # 384 linked at their 5′ ends (e.g., 3′-CCACGAGTGAGCGAAACCC-5′-X-5′-CCCAAAGCGAGTGAGCACC-3′, wherein X represents a non-nucleotidic linker). Alternatively, a 3GA compound comprising two different oligonucleotides such as Oligo # 385 and Oligo # 386 (e.g., 3′-ACCGGACGAGAAGGGTACA-5′-X-5′-GGTGGAGTGAGGCTGGTGC-3′, wherein X represents a non-nucleotidic linker) will be referred to herein, for example, as “3GA 385/386”.

Certain embodiments provide gene silencing compounds comprising two oligonucleotides independently selected from the oligonucleotides listed in Table 3. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, or combinations thereof. In certain embodiments, the oligonucleotides of the gene silencing compound are the same. In certain embodiments, the oligonucleotides of the gene silencing compounds are different.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 80% complimentary to its target site with SEQ ID NO: 387, SEQ ID NO: 388,

SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, or SEQ ID NO: 406. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 85% complimentary to its target site with SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, or SEQ ID NO: 406. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 90% complimentary to its target site with SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, or SEQ ID NO: 406. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 95% complimentary to its target site with SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, or SEQ ID NO: 406.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 49, 50, 51, 52, 53, 54, 75, 76, 77, 78, 79, 80, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, or 365, and is at least 80% complimentary to its target site with SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401, SEQ ID NO: 403, or SEQ ID NO: 405. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 49, 50, 51, 52, 53, 54, 75, 76, 77, 78, 79, 80,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, or 365, and is at least 85% complimentary to its target site with SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401, SEQ ID NO: 403, or SEQ ID NO: 405. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 49, 50, 51, 52, 53, 54, 75, 76, 77, 78, 79, 80,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, or 365, and is at least 90% complimentary to its target site with SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401, SEQ ID NO: 403, or SEQ ID NO: 405. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 49, 50, 51, 52, 53, 54, 75, 76, 77, 78, 79, 80,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, or 365, and is at least 95% complimentary to its target site with SEQ ID NO: 387, SEQ ID NO: 389, SEQ ID NO: 391, SEQ ID NO: 393, SEQ ID NO: 395, SEQ ID NO: 397, SEQ ID NO: 399, SEQ ID NO: 401, SEQ ID NO: 403, or SEQ ID NO: 405.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 80% complimentary to its target site within SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 392, SEQ ID NO: 394, SEQ ID NO: 396, SEQ ID NO: 398, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, or SEQ ID NO: 406. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 85% complimentary to its target site within SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 392, SEQ ID NO: 394, SEQ ID NO: 396, SEQ ID NO: 398, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, or SEQ ID NO: 406. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 90% complimentary to its target site within SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 392, SEQ ID NO: 394, SEQ ID NO: 396, SEQ ID NO: 398, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, or SEQ ID NO: 406. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 95% complimentary to its target site within SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 392, SEQ ID NO: 394, SEQ ID NO: 396, SEQ ID NO: 398, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, or SEQ ID NO: 406.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, and is at least 80% complimentary to SEQ ID NO: 387. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, and is at least 85% complimentary to SEQ ID NO: 387. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, and is at least 90% complimentary to SEQ ID NO: 387. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, or 8, and is at least 95% complimentary to SEQ ID NO: 387.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and is at least 80% complimentary to SEQ ID NO: 388. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and is at least 85% complimentary to SEQ ID NO: 388. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and is at least 90% complimentary to SEQ ID NO: 388. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and is at least 95% complimentary to SEQ ID NO: 388.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 49, 50, 51, 52, 53, 54, and is at least 80% complimentary to SEQ ID NO: 389. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 49, 50, 51, 52, 53, 54, and is at least 85% complimentary to SEQ ID NO: 389. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 49, 50, 51, 52, 53, 54, and is at least 90% complimentary to SEQ ID NO: 389. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 49, 50, 51, 52, 53, 54, and is at least 95% complimentary to SEQ ID NO: 389.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and is at least 80% complimentary to SEQ ID NO: 390. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and is at least 85% complimentary to SEQ ID NO: 390. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and is at least 90% complimentary to SEQ ID NO: 390. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, and is at least 95% complimentary to SEQ ID NO: 390.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 75, 76, 77, 78, 79, 80, and is at least 80% complimentary to SEQ ID NO: 391. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 75, 76, 77, 78, 79, 80, and is at least 85% complimentary to SEQ ID NO: 391. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 75, 76, 77, 78, 79, 80, and is at least 90% complimentary to SEQ ID NO: 391. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 75, 76, 77, 78, 79, 80, and is at least 95% complimentary to SEQ ID NO: 391.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, and is at least 80% complimentary to SEQ ID NO: 392. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, and is at least 85% complimentary to SEQ ID NO: 392. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, and is at least 90% complimentary to SEQ ID NO: 392. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, and is at least 95% complimentary to SEQ ID NO: 392.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, and is at least 80% complimentary to SEQ ID NO: 393. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, and is at least 85% complimentary to SEQ ID NO: 393. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, and is at least 90% complimentary to SEQ ID NO: 393. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, and is at least 95% complimentary to SEQ ID NO: 393.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, and is at least 80% complimentary to SEQ ID NO: 394. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, and is at least 85% complimentary to SEQ ID NO: 394. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, and is at least 90% complimentary to SEQ ID NO: 394. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, and is at least 95% complimentary to SEQ ID NO: 394.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, and is at least 80% complimentary to SEQ ID NO: 395. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, and is at least 85% complimentary to SEQ ID NO: 395. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, and is at least 90% complimentary to SEQ ID NO: 395. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, and is at least 95% complimentary to SEQ ID NO: 395.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, and is at least 80% complimentary to SEQ ID NO: 396. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, and is at least 85% complimentary to SEQ ID NO: 396. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, and is at least 90% complimentary to SEQ ID NO: 396. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, and is at least 95% complimentary to SEQ ID NO: 396.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, and is at least 80% complimentary to SEQ ID NO: 397. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, and is at least 85% complimentary to SEQ ID NO: 397. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, and is at least 90% complimentary to SEQ ID NO: 397. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, and is at least 95% complimentary to SEQ ID NO: 397.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, and is at least 80% complimentary to SEQ ID NO: 398. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, and is at least 85% complimentary to SEQ ID NO: 398. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, and is at least 90% complimentary to SEQ ID NO: 398. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, and is at least 95% complimentary to SEQ ID NO: 398.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, and is at least 80% complimentary to SEQ ID NO: 399. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, and is at least 85% complimentary to SEQ ID NO: 399. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, and is at least 90% complimentary to SEQ ID NO: 399. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, and is at least 95% complimentary to SEQ ID NO: 399.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, and is at least 80% complimentary to SEQ ID NO: 400. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, and is at least 85% complimentary to SEQ ID NO: 400. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, and is at least 90% complimentary to SEQ ID NO: 400. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, and is at least 95% complimentary to SEQ ID NO: 400.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, and is at least 80% complimentary to SEQ ID NO: 401. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, and is at least 85% complimentary to SEQ ID NO: 401. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, and is at least 90% complimentary to SEQ ID NO: 401. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, and is at least 95% complimentary to SEQ ID NO: 401.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, and is at least 80% complimentary to SEQ ID NO: 402. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, and is at least 85% complimentary to SEQ ID NO: 402. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, and is at least 90% complimentary to SEQ ID NO: 402. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, and is at least 95% complimentary to SEQ ID NO: 402.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, and is at least 80% complimentary to SEQ ID NO: 403. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, and is at least 85% complimentary to SEQ ID NO: 403. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, and is at least 90% complimentary to SEQ ID NO: 403. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, and is at least 95% complimentary to SEQ ID NO: 403.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, and is at least 80% complimentary to SEQ ID NO: 404. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, and is at least 85% complimentary to SEQ ID NO: 404. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, and is at least 90% complimentary to SEQ ID NO: 404. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, and is at least 95% complimentary to SEQ ID NO: 404.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, and is at least 80% complimentary to SEQ ID NO: 405. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, and is at least 85% complimentary to SEQ ID NO: 405. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, and is at least 90% complimentary to SEQ ID NO: 405. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, and is at least 95% complimentary to SEQ ID NO: 405.

In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 80% complimentary to SEQ ID NO: 406. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 85% complimentary to SEQ ID NO: 406. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 90% complimentary to SEQ ID NO: 406. In certain embodiments, the gene silencing compounds comprise two oligonucleotides each, independently, comprising a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 95% complimentary to SEQ ID NO: 406.

In certain embodiments, the invention provides a composition comprising a 3GA compound according to the invention and one or more vaccines, antigens, antibodies, cytotoxic agents, chemotherapeutic agents (both traditional chemotherapy and modern targeted therapies), kinase inhibitors, allergens, antibiotics, agonist, antagonist, antisense oligonucleotides, ribozymes, RNAi molecules, siRNA molecules, miRNA molecules, aptamers, proteins, gene therapy vectors, DNA vaccines, adjuvants, co-stimulatory molecules or combinations thereof.

In certain embodiments, the invention provides a method for inhibiting PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA or protein expression, the method comprising contacting a cell with a gene silencing compound according to the invention. In certain embodiments, the cell can be contacted with two or more gene silencing compounds targeting different regions of the same checkpoint. In certain embodiments, the cell can be contacted with two or more gene silencing compounds targeting different checkpoints.

Certain embodiments further provide a method to reduce PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or or OX40L mRNA or protein expression in an animal comprising administering to the animal a gene silencing compound or composition as described herein to reduce PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L mRNA or protein expression in the animal. In certain embodiments, the animal is a human. In certain embodiments, reducing PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L mRNA or protein expression prevents, treats, ameliorates, or slows progression of disease. In certain embodiments reducing PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L mRNA or protein expression inhibits immune system tolerance. In certain embodiments two or more gene silencing compounds targeting different regions of the same checkpoint can be administered. In certain embodiments two or more gene silencing compounds targeting different checkpoints can be administered.

In certain embodiments provided are methods for inhibiting immune system tolerance to tumors comprising administering to the animal a gene silencing compound or composition as described herein to reduce PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA or protein expression in the animal. In certain embodiments, the animal is a human. In certain embodiments, the gene silencing compound or composition as described herein is administered intratumorally. Thus, the inhibition of PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L mRNA or protein expression may provide a potentially useful immunotherapy strategy for patients with cancer. In certain embodiments two or more gene silencing compounds targeting different regions of the same checkpoint can be administered. In certain embodiments two or more gene silencing compounds targeting different checkpoints can be administered.

In certain embodiments provided are methods for preventing tumor growth and tumor volume. In certain embodiments provided are methods for reducing tumor growth and tumor volume.

In certain embodiments provided are methods, compounds, and compositions for the treatment, prevention, or amelioration of diseases, disorders, and conditions associated with PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L in an individual in need thereof Also contemplated are methods and compounds for the preparation of a medicament for the treatment, prevention, or amelioration of a disease, disorder, or condition associated with PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L. In certain embodiments two or more gene silencing compounds targeting different regions of the same checkpoint can be administered. In certain embodiments two or more gene silencing compounds targeting different checkpoints can be administered.

PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L associated diseases, disorders, and conditions include hyperproliferative diseases, e.g., cancer, carcinomas, sarcomas, lymphomas, and leukemias as well as associated malignancies and metastases. PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L associated diseases, disorders, and conditions can also include autoimmune diseases and disorders.

In certain embodiments provided are PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L gene silencing compounds for use in treating, preventing, or ameliorating a PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L associated disease. In certain embodiments, PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L gene silencing compounds are capable of inhibiting the expression of PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L mRNA and/or PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L protein in a cell, tissue, or animal.

Certain embodiments provide methods comprising administering to an animal a gene silencing compounds as described herein. In certain embodiments two or more gene silencing compounds targeting different regions of the same checkpoint can be administered. In certain embodiments two or more gene silencing compounds targeting different checkpoints can be administered.

Also provided are methods and gene silencing compounds for the preparation of a medicament for the treatment, prevention, or amelioration of disease.

Certain embodiments provide the use of gene silencing compounds as described herein in the manufacture of a medicament for treating, ameliorating, or preventing disease.

Certain embodiments provide gene silencing compounds as described herein for use in treating, preventing, or ameliorating disease as described herein by combination therapy with an additional agent or therapy as described herein. Agents or therapies can be co-administered or administered concomitantly.

Certain embodiments provide the use of a gene silencing compound as described herein in the manufacture of a medicament for treating, preventing, or ameliorating disease as described herein by combination therapy with an additional agent or therapy as described herein. Agents or therapies can be co-administered or administered concomitantly.

Certain embodiments provide the use of a gene silencing compound as described herein in the manufacture of a medicament for treating, preventing, or ameliorating disease as described herein in a patient who is subsequently administered an additional agent or therapy as described herein.

In any of the methods according to the invention, the gene silencing compound according to the invention can variously act by producing direct gene expression modulation effects alone and/or in combination with any other agent useful for treating or preventing the disease or condition that does not diminish the gene expression modulation effect of the gene silencing compound according to the invention. In any of the methods according to the invention, the agent(s) useful for treating or preventing the disease or condition includes, but is not limited to, vaccines, antigens, antibodies, preferably monoclonal antibodies, cytotoxic agents, kinase inhibitors, allergens, antibiotics, siRNA molecules, antisense oligonucleotides, TLR antagonist (e.g. antagonists of TLR3 and/or TLR7 and/or antagonists of TLR8 and/or antagonists of TLR9), chemotherapeutic agents (both traditional chemotherapy and modern targeted therapies), targeted therapeutic agents, activated cells, peptides, proteins, gene therapy vectors, peptide vaccines, protein vaccines, DNA vaccines, adjuvants, and co-stimulatory molecules (e.g. cytokines, chemokines, protein ligands, trans-activating factors, peptides or peptides comprising modified amino acids), or combinations thereof. For example, in the treatment of cancer, it is contemplated that the oligonucleotide-based compound according to the invention may be administered in combination with one or more chemotherapeutic compound, targeted therapeutic agent and/or monoclonal antibody. Alternatively, the agent can include DNA vectors encoding for antigen or allergen. Alternatively, the gene silencing compound according to the invention can be administered in combination with other compounds (for example lipids or liposomes) to enhance the specificity or magnitude of the gene expression modulation of the oligonucleotide-based compound according to the invention.

In any of the methods according to the invention, administration of gene silencing compounds according to the invention, alone or in combination with any other agent, can be by any suitable route, including, without limitation, parenteral, mucosal, oral, sublingual, intratumoral, transdermal, topical, inhalation, intrathecal, intranasal, aerosol, intraocular, intratracheal, intrarectal, vaginal, by gene gun, dermal patch or in eye drop or mouthwash form. In any of the methods according to the invention, administration of gene silencing compounds according to the invention, alone or in combination with any other agent, can be directly to a tissue or organ such as, but not limited to, the bladder, liver, lung, kidney or lung. In certain embodiments, administration of gene silencing compounds according to the invention, alone or in combination with any other agent, is by intratumoral administration. In certain embodiments, administration of gene silencing compounds according to the invention, alone or in combination with any other agent, is by mucosal administration. In certain embodiments, administration of gene silencing compounds according to the invention, alone or in combination with any other agent, is by oral administration. In certain embodiments, administration of gene silencing compounds according to the invention, alone or in combination with any other agent, is by intrarectal administration. In certain embodiments, administration of gene silencing compounds according to the invention, alone or in combination with any other agent, is by intrathecal administration. In certain embodiments, administration of gene silencing compounds according to the invention, alone or in combination with any other agent, is directly to the bladder. In certain embodiments, administration of gene silencing compounds according to the invention, alone or in combination with any other agent, is directly to the lung.

Administration of the therapeutic compositions of gene silencing compounds according to the invention can be carried out using known procedures using an effective amount and for periods of time effective to reduce symptoms or surrogate markers of the disease. For example, an effective amount of a gene silencing compound according to the invention for treating a disease and/or disorder could be that amount necessary to alleviate or reduce the symptoms, or delay or ameliorate the disease and/or disorder. In the context of administering a composition that modulates gene expression, an effective amount of a gene silencing compound according to the invention is an amount sufficient to achieve the desired modulation as compared to the gene expression in the absence of the gene silencing compound according to the invention. The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition. One of ordinary skill in the art can empirically determine the effective amount of a particular compound without necessitating undue experimentation.

When administered systemically, the therapeutic composition is preferably administered at a sufficient dosage to attain a blood level of gene silencing compound according to the invention from about 0.0001 micromolar to about 10 micromolar. For localized administration, much lower concentrations than this may be effective, and much higher concentrations may be tolerated. Preferably, a total dosage of gene silencing compound according to the invention ranges from about 0.001 mg per patient per day to about 200 mg per kg body weight per day. In certain embodiments, the total dosage may be 0.08, 0.16, 0.32, 0.48, 0.32, 0.64, 1, 10 or 30 mg/kg body weight administered daily, twice weekly or weekly. It may be desirable to administer simultaneously, or sequentially a therapeutically effective amount of one or more of the therapeutic compositions of the invention to an individual as a single treatment episode.

The methods according to this aspect of the invention are useful for model studies of gene expression. The methods are also useful for the prophylactic or therapeutic treatment of human or animal disease. For example, the methods are useful for pediatric and veterinary inhibition of gene expression applications.

The examples below are intended to further illustrate certain preferred embodiments of the invention, and are not intended to limit the scope of the invention.

Example 1 Preparation of Oligonucleotide-Based Compounds

The oligonucleotide-based compounds of the invention were chemically synthesized using phosphoramidite chemistry on automated DNA/RNA synthesizer. TAC protected (Except U) 2′-O-TBDMS RNA monomers, A, G, C and U, were purchased from Sigma-Aldrich. 7-deaza-G, inosine and loxoribine monomers were purchased from ChemGenes Corporation. 0.25M 5-ethylthio-1H-tetrazole, PAC-anhydride Cap A and Cap B were purchased from Glen Research. 3% trichloroacetic acid (TCA) in dichloromethane (DCM) and 5% 3H-1,2-Benzodithiole-3-one-1,1-dioxide (Beaucage reagent) were made in house.

Oligonucleotide-based compounds of the invention were synthesized at 1-2 μM scale using a standard RNA synthesis protocol.

Cleavage and Base Deprotection

Oligonucleotide-based compounds of the invention were cleaved from solid support and the solution was further heated at 65° C. to removing protecting groups of exo cyclic-amines. The resulting solution was dried completely in a SpeedVac.

IE HPLC Purification

Oligonucleotide-based compounds of the invention were purified by ion exchange HPLC.

Column: Dionex DNAPac 100 column (22×250)

Column Heater: ChromTech TL-105 HPLC column heater, temperature is set to 80° C.

Buffer A: 20 mM Tris-HCl, pH 7.0, 20% acetinitrile

Buffer B: 3.0 M NaCl, 20 mM Tris-HCl, pH 7.0, 20% acetonitrile

Flow rate: 10 ml/min

Gradient:

-   -   0-2 min: 0% B     -   2-11 min: 0% B to 35% B     -   11-41 min: 35% B to 90% B     -   41-45 min: 100% B

Crude solution of oligonucleotide-based compounds of the invention was injected into HPLC. Above gradient is performed and the fractions were collected. All fractions containing more than 90% desired product were mixed, and then the solution was concentrated to almost dry by RotoVap. RNAse-free water was added to make final volume of 10 ml.

C-18 Reversed Phase Desalting

CC-18 Sep-Pak cartridge purchased from Waters was first conditioned with 10 ml of acetonitrile followed by 10 ml of 0.5 M sodium acetate. 10 ml of the solution of oligonucleotide-based compounds of the invention was loaded. 15 ml of water was then used to wash out the salt. The oligonucleotide-based compounds of the invention was eluted out by 1 ml of 50% acetonitrile in water.

The solution is placed in SpeedVac for 30 minutes. The remaining solution was filter through a 0.2 micro filter and then was lyophilized to dryness. The solid was then re-dissolved in water to make the desired concentration.

The final solution was stored below 0° C.

Capillary Electrophoresis

Oligonucleotide-based compounds of the invention were analyzed by capillary electrophoresis according to the following conditions.

Instrument: Beckman 5010

Capillary: 62 cm ssDNA capillary

Sample preparation: 0.2 OD of oligonucleotide-based composition according to the invention was dissolved in 200ul of RNAse-free water.

Injection: electro-kinetic injection at 5KV for 5 seconds.

Running condition: 14KV for 50 minutes at 30° C.

Ion Exchange HPLC Analysis

Oligonucleotide-based compounds of the invention were analyzed by ion exchange HPLC according to the following conditions:

Column: Dionex DNAPac guard column (22×250)

Column Heater: ChromTech TL-105 HPLC column heater, temperature is set to 80° C.

Buffer A: 100 mM Tris-HCl, pH 8.0, 20% acetinitrile

Buffer B: 2.0 M LiCl, 100 mM Tris-HCl, pH 8.0, 20% acetonitrile

Flow rate: 2 ml/min

Gradient:

-   -   0-2 min: 0% B     -   2-10 min: 0% B to 100% B     -   10-15 min: 100% B

PAGE Analysis

0.3 OD of oligonucleotide-based compounds of the invention was loaded on 20% polyacrylamide gel and was running at constant power of 4 watts for approximately 5 hours. The gel was viewed under short wavelength UV light.

Dual Luciferase Reporter System Assay

Hepa 1-6 cells are co-transfected with GSO and target plasmid simultaneously using LIPOFECTAMINE® 2000 on day one (20,000 c/well). RLuc siRNA was used as the positive control and GSO mu/hu universal control was used as the negative control. On day two (24 hours post-transfection), luminescence measurements for both reporter genes are taken separately: Firefly luciferase: expression serves as the normalizer for the assay; Renilla luciferase: substrate includes a “stop” reagent to quench luminescence from firefly. Separate luminescence measurements are taken to correspond to renilla-target transcript expression. Substrate includes DTT to lyse cells. Results are shown in Table 4A and Table 4B.

TABLE 4A % KD Luciferase 3GA Screen # GSO sequence (25 nM)  1 3′-GACCGTACACCCAGGCCG-5′-X- 72.80 5′-GCCGGACCCACATGCCCAG-3′  2 3′-ACACCCAGGCCGTCCATGG-5′-X- 74.00 5′-GGTACCTGCCGGACCCACA-3′  3 3′-TCGACCGTTAGTCCCACCG-5′-X- 73.10 5′-GCCACCCTGATTGCCAGCT-3′  4 3′-TCTCCCTCGTTTACGGTGG-5′-X- 62.60 5′-GGTGGCATTTGCTCCCTCT-3′  5 3′-TCCCTGCTCTCTTCTGTGG-5′-X- 20.80 5′-GGTGTCTTCTCTCGTCCCT-3′  6 3′-ACCCTGCATCCCCGAGTCG-5′-X- 52.80 5′-GCTGAGCCCCTACGTCCCA-3′  7 3′-TGTTCCACGTCTCGACCCC-5′-X- 76.50 5′-CCCCAGCTCTGCACCTTGT-3′  8 3′-TCCCTTGGTCGTCTCGATC-5′-X- 71.50 5′-CTAGCTCTGCTGGTTCCCT-3′  9 3′-TACGTCTAGGGTGTCCGCG-5′-X- 76.32 5′-GCGCCTGTGGGATCTGCAT-3′ 10 3′-CACGATGTTGACCCGACCG-5′-X- 80.51 5′-GCCAGCCCAGTTGTAGCAC-3′ 11 3′-TCGTTGGTCTGCCTGTTCG-5′-X- 64.70 5′-GCTTGTCCGTCTGGTTGCT-3′ 12 3′-TCCCACTGTCTCTCTTCCC-5′-X- 63.00 5′-CCCTTCTCTCTGTCACCCT-3′ 13 3′-CCCACTGTCTCTCTTCCCG-5′-X- 71.30 5′-GCCCTTCTCTCTGTCACCC-3′ 14 3′-CCACTGTCTCTCTTCCCGT-5′-X- 61.91 5′-TGCCCTTCTCTCTGTCACC-3′ 15 3′-ACGATCAGACCCAGGACCG-5′-X- 53.22 5′-GCCAGGACCCAGACTAGCA-3′ 16 3′-TCAGACCCAGGACCGGCAG-5′-X- 73.38 5′-GACGGCCAGGACCCAGACT-3′ 17 3′-GTGACTCCGGACTCCTACC-5′-X- 53.02 5′-CCATCCTCAGGCCTCAGTG-3′ 18 3′-GACTCCGGACTCCTACCTG-5′-X- 51.62 5′-GTCCATCCTCAGGCCTCAG-3′ 19 3′-CTCCGGACTCCTACCTGTG-5′-X- 56.13 5′-GTGTCCATCCTCAGGCCTC-3′ 20 3′CCGGACTCCTACCTGTGAC-5′-X- 46.22 5′-CAGTGTCCATCCTCAGGCC-3′ 21 3′-CCTCTTCGTCCGTCCCACG-5′-X- 68.62 5′-GCACCCTGCCTGCTTCTCC-3′ 22 3′-TCTTCGTCCGTCCCACGTC-5′-X- 73.88 5′-CTGCACCCTGCCTGCTTCT-3′ 23 3′-GGGTCCGTCGTCCACAGTG-5′-X- 61.92 5′-GTGACACCTGCTGCCTGGG-3′ 24 3′-GGATGTCCCTCCCGGTCTA-5′-X- 40.93 5′-ATCTGGCCCTCCCTGTAGG-3′ 25 3′-ATGTCCCTCCCGGTCTACG-5′-X- 41.82 5′-GCATCTGGCCCTCCCTGTA-3′ 26 3′-GTCCCTCCCGGTCTACGTC-5′-X- 57.94 5′-CTGCATCTGGCCCTCCCTG-3′ 27 3′-CCCTCCCGGTCTACGTCAG-5′-X- 58.14 5′-GACTGCATCTGGCCCTCCC-3′ 28 3′-CTCCCGGTCTACGTCAGTG-5′-X- 63.13 5′-GTGACTGCATCTGGCCCTC-3′ 29 3′-GGTTCTGACCCGTGTCCTC-5′-X- 59.63 5′-CTCCTGTGCCCAGTCTTGG-3′ 30 3′-TCGAGGACCGACACCACCC-5′-X- 77.64 5′-CCCACCACAGCCAGGAGCT-3′ 31 3′-CGAGGACCGACACCACCCG-5′-X- 80.53 5′-GCCCACCACAGCCAGGAGC-3′ 32 3′-GGGTCCGTCGTGGAGTCCG-5′-X- 62.62 5′-GCCTGAGGTGCTGCCTGGG-3′ 33 3′-CCGTCCCTTCGACTCCGTC-5′-X- 73.27 5′-CTGCCTCAGCTTCCCTGCC-3′ 34 3′-CGTCCCTTCGACTCCGTCA-5′-X- 78.22 5′-ACTGCCTCAGCTTCCCTGC-3′ 35 3′-CCCGTCCGTCTCGACCTCC-5′-X- 60.58 5′-CCTCCAGCTCTGCCTGCCC-3′ 36 3′-CCGTCCGTCTCGACCTCCG-5′-X- 54.52 5′-GCCTCCAGCTCTGCCTGCC-3′ 37 3′-CAGGGTTCCAGTCCTCCCG-5′-X- 71.63 5′-GCCCTCCTGACCTTGGGAC-3′ 38 3′-GGGTTCCAGTCCTCCCGTC-5′-X- 69.94 5′-CTGCCCTCCTGACCTTGGG-3′ 39 3′-GTTCCAGTCCTCCCGTCCC-5′-X- 71.58 5′-CCCTGCCCTCCTGACCTTG-3′ 40 3′-GTCCCGGACCCACCCTTCC-5′-X- 57.15 5′-CCTTCCCACCCAGGCCCTG-3′ 41 3′-CCCGGACCCACCCTTCCAT-5′-X- 51.93 5′-TACCTTCCCACCCAGGCCC-3′ 42 3′-CGGACCCACCCTTCCATGT-5′-X- 31.04 5′-TGTACCTTCCCACCCAGGC-3′ 43 3′GACCCACCCTTCCATGTCC-5′-X- 44.89 5′-CCTGTACCTTCCCACCCAG-3′ 44 3′-GTCCCGGTGGTCGTAGGTC-5′-X- 61.72 5′-CTGGATGCTGGTGGCCCTG-3′ 45 3′-TCCCGGTGGTCGTAGGTCC-5′-X- 52.12 5′-CCTGGATGCTGGTGGCCCT-3′ 46 3′TCCGTGGACTCACCGACCC-5′-X- 76.87 5′-CCCAGCCACTCAGGTGCCT-3′ 47 3′-CTCACCGACCCTGTTCCCT-5′-X- 68.50 5′-TCCCTTGTCCCAGCCACTC-3′ 48 3′-CACCGACCCTGTTCCCTAG-5′-X- 74.30 5′-GATCCCTTGTCCCAGCCAC-3′ 49 3′-GCCCTCGACCTGGACGAAC-5′-X- 67.80 5′-CAAGCAGGTCCAGCTCCCG-3′ 50 3′-TGTCGTTGAAGTCCCCCTC-5′-X- 76.40 5′-CTCCCCCTGAAGTTGCTGT-3′ 51 3′-TGCCACCACGCCTGATGTT-5′-X- 84.40 5′-TTGTAGTCCGCACCACCGT-3′ 52 3′-TCCCTTGTGTCTCCAGTGG-5′-X- 58.30 5′-GGTGACCTCTGTGTTCCCT-3′ 53 3′-ATCCCTCCGTCTCCGTCCG-5′-X- 76.10 5′-GCCTGCCTCTGCCTCCCTA-3′ 54 3′-GACTTCCTTGTCCGACCCG-5′-X- 70.30 5′-GCCCAGCCTGTTCCTTCAG-3′ Where X is glycerol

TABLE 4B % KD Luciferase Screen 3GA # (25 nM) 55 72.55 56 16.18 57 68.59 58 82.08 59 64.04 60 61.19 61 55.65 62 29.88 63 44.00 64 73.27 65 69.04 66 76.39 67 67.30 68 84.30 69 61.65 70 59.28 71 60.44 72 49.61 73 65.21 74 52.34 75 82.43 76 68.25 77 83.97 78 82.25 79 67.84 80 41.54 81 80.09 82 53.95 83 74.05 84 2.78 85 53.89 86 53.70 87 34.15 88 77.07 89 23.27 90 41.99 91 41.36 92 60.45 93 58.99 94 74.51 95 10.33 96 9.46 97 42.36 98 27.05 99 24.30 100 10.54 101 85.55 102 60.69 103 63.04 104 59.83 105 57.80 106 71.35 107 74.39 108 74.04 109 80.27 110 89.98 111 86.33 112 88.35 113 84.67 114 57.13 115 56.03 116 77.36 117 72.63 118 74.24 119 79.93 120 86.42 121 57.13 122 49.63 123 65.62 124 72.63 125 96.00 126 71.45 127 −0.08 128 4.49 129 60.37 130 67.01 131 10.42 132 72.11 133 66.46 134 58.22 135 40.56 136 75.82 137 69.22 138 69.06 139 79.03 140 10.17 141 25.50 142 84.19 143 81.61 144 70.57 145 78.67 146 66.46 147 58.98 148 69.06 149 97.18 150 81.25 151 30.14 152 73.42 153 79.87 154 63.67 155 71.18 156 64.26 157 84.39 158 91.96 159 87.62 160 85.37 161 90.51 162 90.48 163 88.20 164 82.16 165 79.86 166 88.55 167 91.35 168 81.98 169 94.67 170 84.41 171 82.59 172 31.12 173 67.25 174 36.58 175 55.38 176 73.10 177 77.05 178 91.25 179 83.86 180 85.21 181 72.85 182 61.27 183 81.35 184 68.97 185 67.13 186 57.30 187 79.41 188 41.38 189 50.80 190 52.18 191 63.14 192 82.52 193 41.99 194 73.39 195 76.17 196 85.66 197 98.60 198 47.11 199 42.47 200 87.17 201 74.36 202 58.24 203 59.21 204 42.36 205 74.17 206 76.54 207 30.41 208 68.55 209 69.73 210 59.73 211 54.92 212 56.90 213 69.09 214 77.40 215 39.73 216 39.23 217 41.13 218 23.48 219 79.92 220 29.57 221 64.50 222 73.89 223 81.38 224 70.29 225 69.92 226 81.70 227 59.46 228 81.39 229 88.01 230 75.84 231 58.18 232 29.33 233 61.77 234 72.38 235 45.83 236 39.94 237 66.24 238 49.78 239 23.03 240 59.57 241 41.65 242 44.50 243 18.23 244 37.51 245 58.43 246 70.66 247 74.80 248 70.32 249 90.70 250 73.19 251 81.50 252 87.92 253 76.82 254 55.60 255 42.30 256 44.52 257 81.17 258 64.45 259 79.46 260 41.81 261 46.85 262 83.04 263 78.00 264 69.88 265 59.09 266 39.05 267 34.97 268 83.20 269 86.16 270 49.03 271 70.17 272 86.40 273 67.96 274 44.65 275 65.68 276 66.68 277 76.67 278 39.46 279 69.63 280 68.44 281 57.77 282 67.85 283 61.74 284 69.87 285 58.11 286 41.07 287 42.40 288 42.09* 289 61.77* 290 33.46* 291 49.08* 292 43.43* 293 40.08* 294 57.06* 295 87.34* 296 76.54* 297 36.96* 298 96.71* 299 70.53* 300 88.21* 301 76.86* 302 85.21* 303 74.25* 304 70.61* 305 83.52* 306 65.18* 307 84.36* 308 91.72 309 93.78 310 86.49 311 79.67 312 78.18 313 68.73 314 49.06 315 51.92 316 64.80 317 49.86 318 60.88 319 54.14 320 54.27 321 64.20 322 57.54 323 64.73 324 24.73 325 −3.86 326 58.29 327 85.58 328 38.27 329 54.35 330 37.69 331 42.71 332 77.86 333 34.95 334 29.20 335 41.76 336 55.25 337 56.23 338 44.34 339 42.26 340 33.54 341 32.88 342 46.91 343 25.39 344 53.54 345 68.08 346 65.26 347 70.49 348 33.62 349 78.29 350 87.30 351 92.56 352 82.30 353 62.64 354 84.10 355 72.48 356 87.25 357 68.93 358 77.23 359 74.70 360 43.71 361 86.31 362 52.57 363 38.62 364 64.49 365 66.70 366 77.24 367 89.81 368 77.82 369 62.31 370 80.21 371 58.76 372 71.34 373 65.23 374 65.58 375 78.67 376 67.01 377 32.15 378 49.07 379 53.07 380 58.24 381 72.09 382 63.90 383 68.54 384 15.69 385 25.43 386 40.49 For 3GA compounds numbers 55 through 386 listed in Table 4B, glycerol is the non-nucleotidic linker.

Flow Cytometric Analysis

Whole blood samples with anticoagulant EDTA from mice in study were stained for 30 minutes in the dark at room temperature with the following labeled antibodies from BD Biosciences in the presence of mouse Fc blocker (Affymetrix eBioscience, 14-0161): rat anti-mouse CD3-Alexa Fluor 647 (557869), rat anti-mouse CD4-Alexa Fluor 647 (557681), rat anti-mouse CD8-Alexa Fluor 488 (557668) or the corresponding isotype controls. Red blood cells were lysed with freshly prepared 1×RBC lysis buffer (eBioscience, 00-4300) and washed with flow cytometry staining buffer (BD Biosciences, 554657). Resuspended cell suspensions in the flow cytometry staining buffer were run on BD Accuri C6 to acquire data and analyzed by FLOWJO (TreeStar).

IC50 Analysis

Hepa 1-6 cells are co-transfected with 3GA and target plasmid simultaneously using LIPOFECTAMINE® 2000 on day one (20,000 c/well). Concentration of 3GAs were ranging from 0.019 to 41.7 nM with a 3-fold increment. RLuc siRNA was used as the positive control and 3GA mu/hu universal control was used as the negative control. On day two (24 hours post-transfection), luminescence measurements for both reporter genes are taken separately: Firefly luciferase: expression serves as the normalizer for the assay; Renilla luciferase: substrate includes a “stop” reagent to quench luminescence from firefly. Separate luminescence measurements are taken to correspond to renilla-target transcript expression. Substrate includes DTT to lyse cells. IC50 of 3GAs was calculated using GraphPad Prism 6. Results are shown in Table 5.

TABLE 5 3GA Target GSO Sequence IC₅₀ # target Site 5′ to 3′ (nM)  75 mIDO1  138  CTAGCCACAAGGACCCAGG 33.1    81 hIDO1  172  GCAGAGCAAAGCCCACTTC  3.49   92 1239  GCCTCCAGTTCCTTTGGCT  1.53    3 mPD1  115  GCCACCCTGATTGCCAGCT 59.0    10 hPD1  108  GCCAGCCCAGTTGTAGCAC  3.87   33 1591  CTGCCTCAGCTTCCCTGCC  1.57   46 2024  CCCAGCCACTCAGGTGCCT  3.16   54 mPD-L1 3311  GCCCAGCCTGTTCCTTCAG 14.4    55 hPD-L1  571  GGTAGCCCTCAGCCTGACA  5.00   58 1080  CCCACATTGCCTGCATCCC  2.42   64 1855  TCTGTTTGCTTCCTCAGCT  2.51  158 mTIM3 1425  ACTTATCACCCTCCTGCCT  5.55  169 hTIM3   59  CTGCCAGGTCTACAGTCAC 13.8   180 1506  CAGCACCCAGTTTTCCCTA  6.10  183 1716  ATCCCAGCCACTCAGGAGG 32.7   110 mLAG3 1108  GCTCCAGACCCAGAACCTT  6.49  124 hLAG3 1356/ GACAGTGGCATTGAGCTGC 11.9   1369  122 1822/ GCTCCAGCTCCTCTATCTT  9.10  1841  143 1848  GGTTCTTGCTCCAGCTCCT  5.03  195 mCTLA4  563  GTTGCCCATGCCCACAAAG 19.6   225 hCTLA4 1822  GCCTTCTTCTGTCCATGGC  1.71  247 mIDO2 2748  GGTGACTTCCAGGTCTGCA  0.247 249 hID02  156  GGTGTCCATTGCCTTCTGT  2.73  259 1137  AGTCACCACTTTCCTTGCT  3.36  262 1363  GGCTTCCCATGCTTTGCCT  1.28  361 mOX40L 1069  TGGACACCACCCTTTCCAT  0.673 368 hOX40L  712  TGGTCCGTGGTCGTACTTG  0.553 370 1104  CTCCTCGTTCCTCACTTCC  5.01 

In Vivo Mouse Tumor Model

Colon tumor can be implanted in BALB/c mice by subcutaneous injection of 10⁶ CT26.WT cells at right flank (Tumor 1) and 10⁶ CT26.CL25 cells at left flank (Tumor 2) on day 0. Treatment can be initiated on day 6 or when tumor size reached to 70 to 80 mm³ by intra-tumor injection of gene silencing compound according to the invention at various dosages (e.g., 2 mg/kg, 5 mg/kg, 12.5 mg/kg, or 25 mg/kg) on day 6, 10, 13, 16, 20, and 22.

Tumor growth can be monitored twice per week throughout the study period. The study can be terminated with blood, spleen and tumor tissues collected for further evaluation. T lymphocyte population in blood and spleen samples were detected and analyzed by flow cytometry. Spleen IFN-γ-producing cells were detected with ELISPOT assay after culture of spleen cells for 24 hours with tumor antigen beta-gal or AH1 peptide. Tumor tissues were analyzed for gene expression by RT-PCR.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific substances and procedures described herein. For example, antisense oligonucleotides that overlap with the oligonucleotides may be used. Such equivalents are considered to be within the scope of this invention, and are covered by the following claims. 

What is claimed is:
 1. A 3^(rd) generation antisense (3GA) compound comprising two oligonucleotides linked at their 5′ ends, each oligonucleotide, independently, consists of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, or SEQ ID NO:
 406. 2. The 3GA compound according to claim 1, wherein each oligonucleotide, independently, consisting of 12 to 30 nucleotides having a nucleobase sequence comprising a portion of at least 12 contiguous nucleobases complementary to an equal length portion of SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 392, SEQ ID NO: 394, SEQ ID NO: 396, SEQ ID NO: 398, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, or SEQ ID NO:
 406. 3. The 3GA compound according to claim 1, wherein the nucleobase sequence of each oligonucleotide is, independently, at least 90% complementary over its entire length to a nucleobase sequence of SEQ ID NO: 387, 388, 389, 390, 391, 392, 393, 394, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, or
 406. 4. The 3GA compound according to claim 3, wherein the nucleobase sequence of each oligonucleotide is, independently, at least 90% complementary over its entire length to a nucleobase sequence of SEQ ID NO: 388, 390, 392, 394, 396, 398, 400, 402, 404, or
 406. 5. The 3GA compound according to claim 1, wherein each oligonucleotide, independently, comprises a portion of at least 12 contiguous nucleobases of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, or combinations thereof.
 6. The 3GA compound according to claim 5, wherein each oligonucleotide, independently, comprises a portion of least 12 contiguous nucleobases of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 80% complimentary to its target site with SEQ ID NO: 387, SEQ ID NO: 388, SEQ ID NO: 389, SEQ ID NO: 390, SEQ ID NO: 391, SEQ ID NO: 392, SEQ ID NO: 393, SEQ ID NO: 394, SEQ ID NO: 395, SEQ ID NO: 396, SEQ ID NO: 397, SEQ ID NO: 398, SEQ ID NO: 399, SEQ ID NO: 400, SEQ ID NO: 401, SEQ ID NO: 402, SEQ ID NO: 403, SEQ ID NO: 404, SEQ ID NO: 405, or SEQ ID NO:
 406. 7. The 3GA compound according to claim 6, wherein each oligonucleotide, independently, comprises a portion of at least 12 contiguous nucleobases of SEQ ID NOs: 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, 383, 384, 385, or 386, and is at least 80% complimentary to its target site within SEQ ID NO: 388, SEQ ID NO: 390, SEQ ID NO: 392, SEQ ID NO: 394, SEQ ID NO: 396, SEQ ID NO: 398, SEQ ID NO: 400, SEQ ID NO: 402, SEQ ID NO: 404, or SEQ ID NO:
 406. 8. A composition comprising a 3GA compound according to claim 1 and a pharmaceutically acceptable carrier.
 9. The composition according to claim 8, further one or more vaccines, antigens, antibodies, cytotoxic agents, chemotherapeutic agents, kinase inhibitors, allergens, antibiotics, agonist, antagonist, antisense oligonucleotides, ribozymes, RNAi molecules, siRNA molecules, miRNA molecules, aptamers, proteins, gene therapy vectors, DNA vaccines, adjuvants, co-stimulatory molecules or combinations thereof.
 10. A method for inhibiting PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA or protein expression, the method comprising contacting a cell with at least one 3GA compound according to claim
 1. 11. The method according to claim 10, wherein the cell is contacted with two or more 3GA compounds targeting different regions of the same checkpoint.
 12. The method according to claim 10, wherein the cell is contacted with two or more 3GA compounds targeting different checkpoints.
 13. A method for inhibiting immune system tolerance to tumors comprising administering to an animal at least one 3GA compound according to claim 1 or composition according to claim 8 to reduce PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, and/or OX40L mRNA or protein expression.
 14. A method for the treatment of a disease, disorder, or condition associated with PD1, PDL1, IDO1, LAG3, TIM3, CTLA4, IDO2, CEACAM1, OX40, or OX40L in an individual in need thereof, the method comprising administering at least one 3GA compound according to claim
 1. 15. The method according to claim 1, wherein the disease, disorder, or condition is a hyperproliferative disease or an autoimmune disease.
 16. The method according to claim 2, wherein the hyperproliferative disease is cancer, carcinomas, sarcomas, lymphomas, leukemias and associated malignancies and metastases.
 17. The method according to claim 3, wherein the 3GA compound is administered intratumorally. 